Wet Lab Notebook ¶

Shantel A. Martinez | Update 2019.04.05¶

This Wet Lab Notebook contains notes, data, and protocols for wet lab experiments that are not included in the other specific notebooks. When topics overlap, links to other notebooks were added for reference.

TABLE OF CONTENTS:
       Dry Weight Embryo Preliminary Experiment
       2018 Parental Germination Assay Field GH
       2018 Progeny Germination Assay Field
Hormone Project
              Tissue Harvest
              Detailed Notes Week 1, Week2, Week3
              LCMS Run Set Info
              **Need to update the [germination assay](#Germ_Pheno) section **

General Lab Protocols ...
       Basic Autoclaving
       Agrobacterium Transformation
       Hormone Extraction
              Dilution Curve
              LCMS Run Instructions
              LCMS Troubleshooting
       Measuring Dry Weight
       Lyophilizer/FreezeDrier
       Germination Assay
       DNA Extraction
       RNA Extraction
**Miscellaneous**
      General Research Experiment Photo Album

Progeny Germination Assays¶

PURPOSE: Phenotype the Cayuga X Caledonia BC1F8 mapping population using germination assays as a suppliment to fine mapping using spike-wetting test scores.
EXPERIMENT DETAILS
Harvested from Helfer 2018
Julian Date Range:
Data excel file is located in CC BC1F7-8_SM.xlsx that includes dates harvested for germination assays, GREENDormHarvDate column, in the Helfer worksheet.
Seed AR: 5 days
Frozen in -20C from July - April
PROTOCOL followed: public html or in notebook with the exception that we use a tea strainer to remove sterilization solution and rinse water instead of a vacuum

20190405
Started the first set of plating assays.
8 lines, thawed 10am, threshed 1pm, and plated 2:30pm the same day.

TOC

Autoclave Protocol ¶

Water, beakers, filter papers, bottles

I've tried to sterilize tips once on the 4th floor of Bradfield autoclave and they were all soaked with water. I either ran the wrong program, I placed the tips in a bad orientation that caught steam, or something, and I have not tried to sterilize tips again.

STEP 1. LIQUIDS Prepare the items for sterilization:

glass bottles with caps containing liquid media, water or buffers - liquid filled no more than container fill-line, capped with cap slightly-loosened one turn to prevent loss, autoclave tape on top of lid.
glass or plastic containers with NO LIDS containing liquid media, liquid media with agar, water or buffers (flasks, beakers, pitchers, etc.) - liquid filled no more than HALF-VOLUME of the container, covered in heavy-duty aluminum foil with no holes and aluminum hanging at least 1 inch over the edge of the opening, autoclave tape on the side of the foil cover overlapping on the glass to secure the foil.

STEP 1. NON-LIQUIDS Prepare the items for sterilization:

glass bottles with caps - washed, air-dried completely, capped with cap half-loosened, autoclave tape on top of lid.
plastic large centrifuge bottles with caps - washed, air-dried completely, capped with cap very loosely covering the opening and NOT tightened, autoclave tape on top of lid.
glass test tubes with caps - washed, air-dried completely, capped firmly, put in non-labeled rack, autoclave tape on front of rack.
plastic pipet reservoirs/basins - washed, air-dried completely, wrapped in heavy-duty aluminum foil with no exposed corners or edges, 1 piece of autoclave tape on the whole batch only.
chunking spatulas or other glass/metal utensils - washed, air-dried completely, wrapped in aluminum foil with no exposed corners or edges, 1 small piece of autoclave tape on each item.
glass or plastic containers with no lids (Flasks, beakers, etc.) - washed, air-dried completely, covered in heavy-duty aluminum foil with no holes and aluminum hanging at least 1 inch over the edge of the opening, autoclave tape on top of the foil cover.

STEP 2. Place all items in autoclave bins - make sure there is enough space between each item inside a bin. Locate and bring with you the pair of orange autoclave gloves.
STEP 3. Use the large grey cart to bring the bins to the autoclave room.
The autoclaves should always be left ON - BUT - sometimes they will be OFF when you are ready to use them. If the autoclave is OFF:

Check on for notes indicating ‘FIXED’ and not ‘BROKEN’. If OK, proceed -otherwise try a different autoclave.
TURN ON the autoclave simply by pushing the ‘ON’ button above the keypad and Program screen. Make sure the door is closed. You will hear the steam start to run inside the jacket.
BEFORE STARTING A CYCLE, make sure the Jacket pressure reads higher than J10P on the program screen (or also look at the pressure gauge above the door). This may take a little longer to start up, but you can proceed to load the autoclave chamber during this time.

STEP 4. If the autoclave is free - proceed to loading:

Turn door open slightly. STEP BACK AND PUT ON THE AUTOCLAVE GLOVES!!
CAUTION!! Step back away from the edges of the door to avoid getting burned by the steam, which may immediately vent.
Wearing gloves, pull out the shelf completely to access it from the side.

NOTE: If another lab’s items are in the autoclave you can take the bins out and put them on any open peripheral bench tops in the room. If the contents of the containers are AGAR Media, make sure to notify the owner lab that you removed these from the autoclave. Any other items (liquid without agar and dry items) are OK to leave on the bench top.
Load your bins onto the shelf. Generally, it is better to put the heavier things on the bottom.
Push the shelf back into the autoclave completely.
Close the door gently but quickly enough that it is completely closed before the auto-lock engages. You should hear the door lock.

STEP 5. Check that the jacket pressure is greater than 10 psi. Select your cycle according to the posted key.

NOTE: Time depends on the autoclave system you are using

Type	sterilization	drying
Liquid Media with agar	45 min	30-45 min
all other Liquids	30 min	30 min
Dry Labware	30 min	15 min

STEP 6. Start a timer or note the start time so that you can be sure to retrieve the items shortly after autoclaving finishes.

NOTE: DO NOT leave anything in the autoclave OVERNIGHT!!!

STEP 7. When the cycle ends, the timers will be near zero. Follow the procedure in Step 4 to open the door, retrieve the items WITH AUTOCLAVE GLOVES!!, and bring them back to the lab.

NOTE: Be sure to close the autoclave door completely before leaving.

TOC

Questions on Transformation protocols¶

particle bombardment (Zemetra) vs agro (alvina & Williamson?)
Alvina, what were the different embryo sizes you tested? 1-4mm? or 0.1-0.4cM
Alvina, the Yuji Ishida 2015 Callus Induction Media was the same as the papers WLS media?
Alvina, your CM4C citation of the Agro protocols 2006 Wan and Layton, I could only find the 2015 book version and I couldnt find a CM4C defined media name, is it the media in the protocol by Medvecka and Harwood 2015 callus induction media?

Alvina Gul Research: CRISPR/Cas9 in Bread Wheat¶

OPTIMIZATION OF	TRANSFORMATION	PROTOCOL
Cultivar	Glenn	Medina
Embryo Size	DONE	DONE
Callus Induction Media	DONE	DONE
Regeneration Media	DONE	DONE
Rooting Media	DONE	ongoing

Ishida et al, 2015; Wan and Layton, 2006; Khanna and Daggard, 2003; CIMMYT Laboratory protocols

Compared
Callus Induction Media: CM4C and Yuji, Gelzan?
Regeneration Media: LSZ, MMS0.2C, CIMMYT (+ 1x, 1.5x, 2x BAP)
Rooting Media: LSF

-	OPTIMIZATION OF	TRANSFORMATION	PROTOCOL
Cultivar	Glenn	Medina	.	Jones et al., 2005
Embryo Size	2mm	2mm	.	0.8-1.5mm
Callus Induction Media	CM4C	CM4C (8wk)	Yuji
Regeneration Media	LSZ, MMS0.2C, CIMMYT	LSZ, MMS0.2C	CIMMYT 1x BAP, LSZ
Rooting Media	ongoing	LSF then 1/2MS	.
Final Recommendation	2mm, CM4C, LSZ, ongoing	2mm, CM4C or Yuji, LSZ, LSF + 1/2MS	.	.

Table A Composition of double-strength culture media. All concentrations are shown double-strength except for the supplements added after pH adjustment and sterilization which are shown at their final concentrations.

Component	Inoculation (/L)	Induction (/L)	RDZ (/L)	RPPT (/L)	R (/L)	CM4C	Yuji	LSZ (/L)	LSF (/L)
-	-	Induction	Regen	Selection	-	Induc	Induc	Regen	Rooting
MS Macro salts (×10)	200 ml	200 ml	200 ml	200 ml	200 ml	-	-	100 mL	100mL
L7 Micro salts (×1000)	2 ml	2 ml	2 ml	2 ml	2 ml	-	-	10mL 100x	10mL 100x
FeNaEDTA (×100)	20 ml	20 ml	20 ml	20 ml	20 ml	-	-	10mL	10mL
MS vitamins (×1000)	2 ml	2 ml	-	-	-	-	-	10mL 100x	10mL 100x
Vitamins/Inositol (×200)	-	-	10 ml	10 ml	10 ml	-	-	-	-
Inositol	200 mg	200 mg	200 mg	200 mg	200 mg	-	-	-	-
IBA (100mg/L)	-	-	-	-	-	-	-	-	2mL
Glutamine	1 g	1 g	-	-	-	-	-	-	-
Casein hydrolysate	200 mg	200 mg	-	-	-	-	-	-	-
MES	3.9 g	3.9 g	-	-	-	-	-	0.5g	0.5g
Glucose	20 g	-	-	-	-	-	-	-	-
Maltose	80 g	80 g	60 g	60 g	60 g	-	-	-	-
Sucrose	-	-	-	-	-	-	-	20g	15g
-	pH adjusted to 5.8 then autoclaved	-	pH adjusted to 5.7 then filter sterilized	-	-	-	-	pH adjusted to 5.8 8g agar then autoclaved	Adjust pH to 5.8 and add 3 g Gelrite. Autoclave
2,4-D	2 mg	0.5 mg	0.1 mg	-	-	-	-	-	-
Picloram	2.0 mg	2.0 mg	-	-	-	-	-	-	-
Acetosyringone	200 μM	-	-	-	-	-	-	-	-
Timentin	-	160 mg	160 mg	160 mg	160 mg	-	-	-	-
Zeatin	-	-	5 mg	-	-	-	-	50mL 100mg/L	-
PPT	-	-	-	2–4 mg	3–4 mg	-	-	-	-
Copper(II) sulfate pentahydrate	-	-	-	-	-	-	-	2.5mg	-
Carabenicillin	-	-	-	-	-	-	-	20g	250mg

TOC

Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat¶

Jones et al., 2005 protocol with SM notes.

Scope and limitations¶

This method was developed for the winter wheat cultivar Florida but with minor modifications has also been used to successfully transform the spring wheat varieties Fielder and Cadenza.

Table 1: Summary of main parameters reported for Agrobacterium-mediated transformation of wheat.

Wheat variety (S – spring) (W – winter)	Explant type	Embryo Axis removed	*Agrobacterium*strain (binary vector)	Inoculation (Co-culture) *rt – room temp	Control of *Agrobacterium*cells	Plant selective agent	Transformation Freq. (%)	No of plants reported	Refs
Bobwhite (S)	IE (age NS*); 1–6 d PCIE; 10–25 d EC	NS*	C58-ABI (pMON18365)	3 h, 23–25°C (2–3 d, 24–26°C)	Carbenicillin (250 mg/l)	G418	1.4–4.3	>100	[17]
Bobwhite (S)	4 d PCIE	NS*	C58-ABI (pMON30139 and others)	15–30 min, 23–25°C (2–3 d, 23–25°C)	Carbenicillin (250–500 mg/l)	Glyphosate	4.4	3354	[16]
Bobwhite (S)	1–6 d PCIE; 8–30 d EC	NS*	C58-ABI (pMON18365)	5–60 min, 23–26°C (2–3 d, 24–26°C)	Carbenicillin (250 mg/l)	G418Paromomycin Glyphosate	4.8–19	154	[18]
Bobwhite (S)	3–6 PCIE	NS*	C58C1 (pPTN155)	45 min – 3 h, 25°C (1–3 d, 25°C)	Ticarcillium; Vancomycin Cefatoxin; (50 mg/l)	G418	0.5–1.5	13	[15]
Cadenza (S) Florida (W)	0–72 h IE	Yes	AGL1 (pAL154/156)	15 min-5 h, rt (1–5 d, 24–25°C rt)	Timentin (160 mg/l)	PPT (L-Phosphinothricin)	0.3–3.3	44	[21]
Fielder (S)	6–9 d PCIE	Yes	AGL0 (pBGX1)	30–60 min rt* (2–3 d, 23–24°C)	Timentin (150 mg/l)	GFP, Bialaphos	1.8	4	[19]
Veery-5 (S)	14 d EC	Yes	LBA4404 (pHK21)	15 min at rt* (1 d 27°C, 2 d 22°C)	Timentin (150 mg/l)	Glufosinate ammonium	1.2–3.9	17	[20]
Vesna (S)	IE (age NS*)	NS*	LBA4404 (pTOK233) AGL1 (pDM805)	15–30 min, (3 d, 27°C)	Cefotaxime (300 mg/l)	PPT (L-Phosphinothricin)	0.13–0.41	6	[45]
Various Chinese varieties (NS*)	EC (age NS*)	NS*	AGL1 (pUNN-2)	30–60 min (2 d, 28°C)	Timentin (150 mg/l)	Paromomycin	3.7–5.9	44	[46]

Growth of donor plants¶

1.1 Sow seeds, 4–5 per 21 cm diameter pot, in compost which contains 75% fine-grade peat, 12% screened sterilised loam, 10% 6 mm screened lime-free grit, 3% medium vermiculite, 2 kg Osmocote Plus/m3 (slow-release fertiliser, 15N/11P/13K plus micronutrients), 0.5 kg PG mix/m3 (14N/16P/18K granular fertiliser plus micronutrients (Petersfield Products, Leicestershire, UK). Although other soil formulations may also be suitable.

1.2 Grow wheat plants in environmentally controlled growth rooms for approximately 11 weeks to provide immature seeds.

1.3 Growth rooms are maintained at 18–20°C day and 14–15°C night temperatures with a relative air humidity of 50–70% under a 16 h photo-period provided by banks of 400 W High Temperature Quartz Iodine lamps (Osram Ltd., Berkshire, UK) which give light intensity ~700 μmolm-2s-1 photosynthetically active radiation (PAR).

1.4 Before transferring to these conditions, winter wheat varieties are vernalised from seed for 8 weeks at 4–5°C with a 12 hour photoperiod provided by 70 W fluorescent lamps giving approximately 150 μmolm-2s-1 PAR at 300 mm from the lights.

1.5 The water is supplied by an automated flooding system, but seedling-stage plants are initially top watered individually for a few weeks [50].

Table 2: Summary of Agrobacterium strains and vectors used to investigate wheat transformation.

*Agrobacteriu*m strain (binary vector)	Chromosomal background	Ti plasmid	Opine classification	Additional *vir* genes on binary or helper plasmids	Binary type	Selectable and scorable marker on T-DNA. (Promoter shown in parentheses)
ABI (pMON18365) [17, 18]	C58	Disarmed pTiC58	Nopaline	pMON18365, none reported	normal-binary	npt II (E35S) GUS (E35S)
C58C1 (pPTN155) [15]	C58	Cured/disarmed?	Nopaline	pPTN155, none reported	normal-binary	npt II (35S) GUS (E35S)
AGL1 (pAL154/156) [21]	C58, RecA	pTiBo542 ΔT-DNA	Succinamopine	pAL154, 15.2 Kb fragment from pTiBo542 [47], pAL156, none	super-binary	bar (Ubi1) GUS (Ubi1)
AGL0 (pBGX1 and pTO134) [19]	C58	pTiBo542 ΔT-DNA	Succinamopine	pBGX1, none reported pTO134, none reported	normal-binary	hpt (35S) gfp(Ubi1)bar (35S) sgfpS65T (35S)
AGL1 (pDM805)	C58, RecA	pTiBo542 ΔT	Succinamopine	pDM805, none reported	normal-binary	bar (Ubi1) GUS (Act1)
LBA4404 (pTOK233) [45]	Ach5	DNA Disarmed pAL4404	Octopine	pTOK233, extra copy of vir B, vir C and vir G from pTiBo542 47, [48]	super-binary	hpt (35S) GUS (35S)
LBA4404 (pHK21) [20]	RecA Ach5	Disarmed pAL4404	Octopine	pHK21, extra copy of vir B, vir C and vir G from pTiBo542 [47]	super-binary	bar (Ubi1) GUS (Ubi1)
AGL1 (pUNN-2) [46]	C58, RecA	pTiBo542 ΔT-DNA	Succinamopine	pUNN-2, none reported	normal-binary	npt II (Ubi1))
ABI (pMON30139 and others) [16]	C58	Disarmed pTiC58	Nopaline	pMON30139, none reported	normal-binary	aro A:CP4 (Act1) aroA:CP4 (e35S+ hsp intron)

Growth and preparation of Agrobacterium cells for inoculation¶

STRAINS: Ach5 (LBA4404), AGL0, AGL1, A281 | C58 (Chrm background), pTiBo542(Ti plasmid)
Ach5 successful when augmented with pHK21, but not standard binary plasmid
As of 2005, there is not enough data to state which vir genes are necessary for optimal transformation per genotype.
virG mutant may improve tranformation efficiency

2.1 Initiate Agrobacterium liquid cultures by adding ~200 μl of a standard glycerol inoculum to 10 ml MG/L [51] (Table 3) plus antibiotics. Prepare as many 10 ml cultures as plates to be treated.

Table 3: Composition of medium MG/L

Component	/litre
Mannitol	5 g
L-Glutamic acid	1 g
KH2PO4	250 mg
NaCl	100 mg
MgSO4·7H2O	100 mg
Tryptone	5 g
Yeast extract pH 7.0	2.5 g
Biotin (added after autoclaving from stock at 1 mg/100 ml (add 100 μl to 1 litre MG/L)	1 μg

2.2 Incubate at 27–29°C, shaking (250 rpm) for 12–24 hours (to reach an OD >1 (Abs = 600 nm)).

2.3 Pellet the Agrobacterium culture at 4500 g for 10 minutes and resuspend in 4 ml single-strength inoculation medium (see 6.2.2) supplemented with 200 μM acetosyringone for each 10 ml culture.

2.4 Replace the cultures back on the shaker until required, but they should be used within 3 hours.

The antibiotics used depend on the selectable markers in the Agrobacterium strain and binary vectors used. For the AGL1 strain used in this protocol, carbenicillin (200 mg/l) is used and pAL154/156 combinations are selected with kanamycin (100 mg/l) which is the selectable marker on pAL156.

Preparation of explants¶

3.1 Ear collection and surface sterilization¶

3.1.1 Collect ears at approximately 12–16 days post-anthesis, a few seeds can be opened at the time of collection to determine the size and texture of the embryos, which should be 0.8 – 1.5 mm in length and translucent in appearance.

3.1.2 Surface sterilise by rinsing in 70% (v/v) aqueous ethanol for 1 minute then 15 minutes in 10% (v/v) Domestos bleach solution (Lever) with gentle shaking. Rinse with sterile distilled water at least three times.

Note, due to asynchronous development, only half or two thirds of the seeds on any one ear will be suitable, the seeds nearest to the peduncle are generally younger and smaller.

3.2 Isolation of immature embryos¶

VARIATIONS: freshly isolated immature embryos, pre-cultured embryos (1-6d), or embryogenic callus derived from immature embryos
PROBLEM: This protocol uses fresh isolated embryos, aproblem with the is precocious zygotic germination, but can be supressed by adding hormones such as dicamba, ABA, high levels 2,4-D to cutlure medium.

3.2.1 Isolate the embryos from the seed under a stereo microscope in a sterile environment using a sharp scalpel.

SIZE: embryos >2mm give higher transient expression but low regen freq compared to <1.5mm. This protocol suggests using 0.8-1.5mm, however I would shoot for 1.5mm to possible get the best of the high expression while having optimal regen freq (but note, that is an unsupported theory).

3.2.2 Remove and discard the embryo axis first then isolate the remaining portion of the embryo which is now referred to as the scutellum.

3.2.3 Plate scutella with the axis side (now removed) down onto semi-solid inoculation medium in 55 mm Petri dishes, about 50 scutella per plate.

3.2.4 It is important to inoculate each plate of 50 scutella with Agrobacterium tumefaciens, as described below, before isolating embryos for the next plate.

Inoculation of scutella with Agrobacterium tumefaciens¶

VARIATIONS: air-drying or osmotic conditioning explants during Agro co-cultivation. Air-drying pre-cultured imm embryos and callus increased T-DNA delivery and suppressed Agro cell growth (facilitated better plant cell recovery), but note this was just on Bobwhite. Osmotic conditions 10% sucrose (increase expression in one study) or 20% maltose prior to Agro innoculation.
One or both these steps are carried out in darkness at approximately 25°C

4.1 Inoculation Take the resuspended Agrobacterium suspension from the shaker, add 60 μl 1% Silwet to make a final concentration of 0.015% and pour the whole 4 ml over a batch of 50 plated scutella.

Silwet is a sufactant. Surfactants increases T-DNA delivery. Conc >0.05% reduced survival and callus formation. 0.04% shown to have positive effect. [21] deteremined 0.01% optimal conc.
QUESTION: did they look at 0.02, 0.03, abd 0.04, and was it bad like 0.05? Silwet has been used at concentrations as high as 0.05% for pre-cultured embryos and calli [15] but this protocol has no pre-culture treatments.

4.2 Incubate for 1–3 hours at room temperature while preparing more scutella for inoculation as described in 3.2.

4.3 Co-Cultivation Transfer the scutella without blotting, keeping the ex-axis side down, onto fresh inoculation medium in 55 mm dishes. Allow to co-cultivate in the dark at 22–23°C for 2–3 days.

200 μM acetosyringone in the Agrobacterium or co-cultivation medium markedly increased T-DNA delivery
Enhanced transient GFP expression was observed in wheat cell clusters with acetosyrigone at 400 μM in the co-cultivation but not the inoculation media

Control of Agrobacterium and induction of embryogenic calli, regeneration and selection¶

Now we need to inhibit the growth of Agrobacterium cells (with antibiotics) and promote regeneration and selection of transformants.
ANTIBIOTICS: Timentin, carbenicillin (both common), cefatoxin, cefotaxime, tiracillium, and vancomycin

5.1 Induction After 2–3 days co-cultivation, transfer all scutella to induction medium (Table 4) and continue to incubate in the dark at 24–25°C.

Table 4 Composition of double-strength culture media. All concentrations are shown double-strength except for the supplements added after pH adjustment and sterilization which are shown at their final concentrations.

Component	Inoculation (/L)	Induction (/L)	RDZ (/L)	RPPT (/L)	R (/L)
MS Macro salts (×10)	200 ml	200 ml	200 ml	200 ml	200 ml
L7 Micro salts (×1000)	2 ml	2 ml	2 ml	2 ml	2 ml
FeNaEDTA (×100)	20 ml	20 ml	20 ml	20 ml	20 ml
MS vitamins (×1000)	2 ml	2 ml	-	-	-
Vitamins/Inositol (×200)	-	-	10 ml	10 ml	10 ml
Inositol	200 mg	200 mg	200 mg	200 mg	200 mg
Glutamine	1 g	1 g	-	-	-
Casein hydrolysate	200 mg	200 mg	-	-	-
MES	3.9 g	3.9 g	-	-	-
Glucose	20 g	-	-	-	-
Maltose	80 g	80 g	60 g	60 g	60 g
-	pH adjusted to 5.8 then autoclaved	-	pH adjusted to 5.7 then filter sterilized	-	-
2,4-D	2 mg	0.5 mg	0.1 mg	-	-
Picloram	2.0 mg	2.0 mg	-	-	-
Acetosyringone	200 μM	-	-	-	-
Timentin	-	160 mg	160 mg	160 mg	160 mg
Zeatin	-	-	5 mg	-	-
PPT	-	-	-	2–4 mg	3–4 mg

5.2 Regeneration After 18 days, transfer embryogenic calli to RDZ medium (Table 4), and incubate at 24–25°C but in the light. Embryogenic calli derived from the same immature embryo should be kept intact without breaking up.

5.3 Selection After 3 weeks, transfer embryogenic calli to selection medium RPPT (or appropriate selection agent, Table 4). At this point, the calli can be broken into defined shoots/roots, but it is important to keep these together, or mark them clearly as there is possibility that these may be clones.

Protocol described in the present paper was optimised for bar/glyphosate selection with a GUS assay to confirm T-DNA integration
Delayed Selection is in this protocol. Selection for plant transformation is often initiated a few days after co-cultivation during the callus-induction phase and maintained during the latter regeneration steps.
VARIATIONS: Antibiotic resistant (hpt (aph4-Ib) or nptII (aph3'II) genes), glufosinate ammonium-based herbicide resistant (bar gene), glyphosate-based herbicide resistant (aroA:CP4 gene)

5.4 Continue transferring to fresh RPPT every 3 weeks until PPT tolerant plantlets are ready to be potted to soil.

Note, at the end of the first round of selection, some of the transgenic plants may be identified by GUS assay on leaf fragments. If they have good strong roots, they may be transferred to soil or put into the vernalization room immediately, otherwise, transfer them to R medium without PPT for root strengthening (Table 4).

Materials¶

6.1 Media for growing Agrobacterium tumefaciens¶

See Table 3.

6.2 Media for plant tissue culture¶

6.2.1 Plant tissue culture media are prepared from stock solutions at double strength to allow the addition of an equal volume of gelling agent; Phytagel for inoculation and induction media, agargel for RDZ, RPPT, and R media. Gelling agents are also prepared at double strength (Phytagel at 4 g/l and agargel at 10 g/l) and autoclaved at 121°C for 20 min (see Table 4).

6.2.2 To make single-strength liquid inoculation media for resuspending Agrobacterium cells in section 2.3, simply mix double-strength medium with autoclaved, distilled water.

Stock solutions for basal culture media¶

Detailed below are the recipes for stock solutions of basal culture media components adapted from [50].

6.2.3 MS Macrosalts (×10):
16.5 g/l NH4NO3 (Fisher Scientific, Leicestershire, UK),
19.0 g/l KNO3 (Sigma-Aldrich, Dorset, UK),
1.7 g/l KH2PO4 (Fisher Scientific UK),
3.7 g/l MgSO4·7H2O (Fisher Scientific UK),
4.4 g/l CaCl2·2H2O (Fisher Scientific UK).

Dissolve each component in distilled water separately before mixing. Autoclave at 121°C for 20 min and store at 4°C.

6.2.4 L7 Microsalts (×1000):
15.0 g/l MnSO4 (Fisher Scientific UK),
5.0 g/l H3BO3 (Fisher Scientific UK),
7.5 g/l ZnSO4·7H2O (Fisher Scientific UK),
0.75 g/l KI (Fisher Scientific UK),
0.25 g/l Na2MoO4·2H2O (VWR International Ltd., Leicestershire, UK),
0.025 g/l CuSO4·5H2O (Fisher Scientific, UK),
0.025 g/l CoCl2·6H2O (Sigma-Aldrich).

MnSO4 may have various hydrated states which will alter the required weight. For MnSO4·H2O, add 17.05 g/l, for MnSO4·4H2O, add 23.22 g/l, for MnSO4·7H2O, add 27.95 g/l. Prepare 100 ml microsalt stock solution at a time. Filter sterilise, and store at 4°C.

6.2.5 MS Vitamins (-Glycine) (×1000):
0.1 g/l Thiamine HCl (Sigma-Aldrich),
0.5 g/l Pyridoxine HCl (Sigma-Aldrich),
0.5 g/l Nicotinic acid (Sigma-Aldrich).

Prepare 100 ml at a time. Filter sterilise, and store at 4°C.

6.2.6 Vitamins/Inositol (×200):
40.0 g/l Myo-Inositol (Sigma-Aldrich),
2.0 g/l Thiamine HCl (Sigma-Aldrich),
0.2 g/l Pyridoxine HCl (Sigma-Aldrich),
0.2 g/l Nicotinic acid (Sigma-Aldrich),
0.2 g/l Ca-Pantothenate (Sigma-Aldrich),
0.2 g/l Ascorbic acid (Sigma-Aldrich).
Filter sterilize and store at -20°C in 10 ml aliquots.

Supplements¶

Acetosyringone (3',5'-Dimethoxy-4'-hydroxyacetophenone) (Aldrich D12,440-6: MW-196.20), Dissolve in 70% ethanol to give 10 mg/ml or 50 mM stock solution. Filter sterilise, aliquot and store at -20°C.
2,4-Dichlorophenoxyacetic acid (2,4-D) (Sigma-Aldrich), 1 mg/ml in ethanol/water (dissolve powder in ethanol then add water to volume). Filter sterilise, and store at -20°C in 1 ml aliquots.
Zeatin mixed isomers (10 mg/ml) (Sigma-Aldrich), Dissolve powder in small volume 1 M HCl and make up to volume with water, mix well/vortex. Filter sterilise, and store at -20°C in 1 ml aliquots.
Picloram (1 mg/ml) (Sigma-Aidrich), Dissolve picloram in water, filter sterilise and store at -20°C in 2 ml aliquots.
Timentin (300 mg/ml) (Melford, UK), Dissolve Timentin (Ticarcillin/Clavulanic (15:1)) in water, filter sterilise and store at -20°C in 1 ml aliquots.
PPT (10 mg/ml)(Glufosinate Ammonium) (Melford, UK), Dissolve in water, mix well/vortex, filter sterilize, and store at -20°C in 1 ml aliquots.
Silwet L-77 (1% v/v) (Lehle seeds, USA), Dissolve in water, filter sterilize, and store at 4°C in 0.5 ml aliquots.

Quality Control¶

Plants escaping selection: a percentage of the number of plants (n) that went through selection AND did not have a successful transformation of the gene of interest against all of the plants (m) prior to screening (n/m) 100
**Regeneration frequencies: number of - lines / total number of Transformation efficiency: number of independent transgenic lines / total number of immature embryos inoculated Transient expression: T-DNA-delivery***:

TOC

**End of Univ of MO Visit Checklist**

Wash all dishes & put away
Refill tips used
Remind Kate about liquid waster container & Empty small chemical bottles
Grab cord & glasses case

**Current To Do List**

Screenshot the equations for the standards on the LCMS Computer.
Call set 3 peaks
Input set 2,3 data into sheet

Hormone Extraction:Learning the Technique Week 1 ¶

2018.09.30 - 2018.10.05

Day 1
Step 0: Make Extraction Solution

At the end we are going to inject 3uL. Step 38 Day 2. Different column in an lcms will change, but for my experiment, we wontchange this 3uL injecction. So our options is to have a final vol of 7uL (dry in speed vac, we resuspend to 7uL) then ass to machine and machine takes 3uL to run. Then we always have 3uL left to rerun if need be.
If we find the peaks are super high, we can change the 7uL to 14uL to make peaks half as high. But this number goes into the final calc, but we need to keep track.
14 ng per 2mL which will lead to a concentration of 7ng/mL (remember, we want to add 2mL into each tube, so 7x2 = 14)
GA12 GA12-ald are not as soluble in aquaous phase, they are more soluble in Acetonitrile, so the peaks usually are low. Sven has found a way to increase the peaks, which we will do to my samples.
We will add double the amount for IS_GA-12ald (24ng/injection) and the others 6x4 = 24ng/injection to be on the safe side. And if we get too high of the samples, then the 2nds set on we can adjust back 6xi = XXng/injection, but we need to keep track for our calculations later.

Prepare one 40mL solution and one 45mL solution of 1st EXTRACTION BUFFER

See PaperLabNotebook table (date 20180930) of volumes used to add the Internal Standards

1:33am 2018.10.01 started 4C incubation.

Day 2
Step 6: I accidently added approx 4mL of the 2nd extraction solution to sample #1018 (Cay_6d_imb) instead of 2mL. This is okay in terms of the extraction, I just had to fill up the #2 tube in step 9 as full as possible, speed vac with all the samples, and then after 1.5 hrs of speedvac, add the remainder of the supernatant from the #1 tube (because I couldnt add it all the first time around in the 5mL tube while still keeping the liquid layer about .75 in away from the top)

Step 10: Make sure not to overfill the tubes, because we need to tip the tubes un-capped into the speedvac

It took us 2.5 hours to get the speedvac working properly. First the rotor wasnt spinnng, somehow after adjusting, turning things on and over, temperatures being reached, it eventually would turn. Then the vacuum wouldnt kick on and pull. We waited for the Vapor trap to reach -104C (90 min), then retried, wouldnt work, let the speedvac reach 45C, still didnt work, check all the hose connections. After getting help from ------- then FINALLY we noticed the vapor traps black crubber connector was town on the edges so the trap wasnt holding vacuum at all. Luckily there was a spare on the shelf, switched the torn rubber connector, and the vacuum held.

With this speed vac, at 20.0 vacuum level, 45C temp, 3hr heat time, 3 hr run time, it only takes 2 hours until the samples are concentrated to a level we can work with (versus svens previous 3hr min note).
4:30pm speedvac checked at 5:45pm

Day 3
Step 16: The water will drip through the column and leave the PVP behind. what we want is the column to be about half full of PVP.
After filling about 10 columns, reshake pvp+water mixture.
It wont hurt if things are too high for the extraction, but it may be a problem when we add our sample, there might not be enough room.
USe the syringe to start the drip, need to press the holder up since you want to form a seal with the syringe, and you dont want to break the holder.

Disposable SPE columns are the ones I set on a rack and use for columns, disposed after 1 use, sometimes refer to as frits or cartridges. But the other column is an LSMC column.
we are doing gravity flow, which makes a big difference compared to a box vacuum or a syringe for each column. The alternative flow techniques dont yield/high quality as well, but they are faster.

For each of the col. steps, be sure to know 1) Am I collecting the flow thru or 2) Am I discarding the flow thru!!! We go back and fourth a lot in this protocol, so keeping track is very important. Svens protocol clearly labels whether we collect or discard, but still pay attention.

Step 17

Accidently mixed in sample 12 with sample 2 on step 17, so I threw out the initial column. created a new column, and added the unmixed remaining sample 2. This will give very low hormone levels, but its better than just omitting this sample all together For sample 12, I was able to catch myself and add the remainder of sample 12 into sample 12 tube during step 17, but it is not the 100% I originally had, I would say I ended up with 350uL/500uL were added to tube 12 step 17

LUNCH

Step 18

Accidently threw away columns thinking we were finished, so we couldnt proceed to step 18. We brought the volume up by adding 2mL K2HPO4 still. We still have the majority of the samples from step 17, we just wont get as much as was possible.
Next time, keep everything until the day is over, no thinking that we can throw tubes away. Then once the day is complete, throw away the earlier steps equipment.

Step 19

added 500ul of hcl to sample 3 instead of 300ul

Day 4
Step 30: left the samples overnight with no heat, since we ran behind in time.
We ran the dilution curve sample in the morning so we were unable to finish the whole extraction.

Day 5
Run LCMS View the notes on for set 1 week 1 in the Run LCMS section

Hormone Extraction: Set 2 Week 2 ¶

2018.10.07 - 2018.10.11
Day 1
Samples 1-19 were used with the ABA-D6 (IS) made previously by S. Nelson in 2017
Samples 20-40 were used with the ABA-D6 (IS) made today y S. Martinez in 2018
Incubated at 11:28pm

Day 2
Start 9:55am
Step 10 started at 1:39pm - finished at 3:44pm (but keep in mind that I spent 11:44 - 1:15pm checking for calculation mistakes)
Done 5:35pm

Day 3
Start 8:50am
Made 500mL of K2HPO4
End 5:38pm speed vac

Day 4
Start 9:00am
Started step 30 speedvac at 10:52am until 12:55pm (I checked t 1.5 hr and there was still about 400uL liquid in the tube)

I noticed the pellets today werent dissolving well in steps 25 & 31, so I asked Sven if you can over vortex the sample and breakdown the hormones? Sven: No. No worries there. So I added another vortex after some time of the initial dissolving, to get the pellet to dissolve even more at both steps 25 & 31.
Even after an hour of "dissolving" all of the samples has a thin yellow flake in them. I added the solution to the columns, like stated in Step 33, but it ma or may not have dissolved properly. If it did not dissolve, why not? If it did dissolve, what is the remaining yellow material left?

5:37pm turned the pump on at a rate of 0.5

Hormone Extraction: Set 3 Week 3 ¶

2018.10.14 - 2018.10.17
Day 1
Samples 1-17 and 18-34 used tube 1 and 2 of 1st extraction buffer, respectively.
Incubated at 5:19pm

Day 2
Start 9:30am
Step 9: spilled Sample 9 into styrofoam well, I re-added liquid in, may be a 'dirty' sample now, but couldnt waste the rep.
Step 10 started at 12:00pm - finished at 2:32pm
Started ON speedvac 4:22pm

Day 3
Start 9:47am
End 7:44pm speed vac

Day 4
Start 9:18am
Step 25 - Samples 19 and 25 had a thin white flake that would not dissolve.

Questions:
Should I be doing standard curves around every run? IF you have to recalibrate the LCMS due to an issue, or the nitrogen feed, then yes. But since I am running all of my samples within 3 weeks, and its a short length. Then I will not need to do another standard curve.
Go over his IS calculations: same except only GA4, & make a new LC running method
How do you know if the LCMS is still running over night? You cant. Try to start the LCMS run earlier in the day, so you are around for most of the work day, but in general, no you cant know when you arent here.
Do we get a retention time x peak data table so I can make a 3x1 graph with endog peaks line graph, IS peaks line graph, difference bar graph? No. We are able to output an image of the plots, but no table with all of the data points.

Additional Lab Comments

Agilent LCMS user and pwd is the same for all Agilent machines and is located in the small drawer below LCMS.
Airtable app has the record of freezer, seed collections, etc.
Slack Group 'Fun with Hormones'
Dont use the columns behind the trash cabinet
Once I pull out last water just, ask Kate to re-order. we will use a lot in the LSMC run.

TOC

LCMS Run Notes & Seq ¶

Set 1 Week 1¶

Run Pameters

Folder	FileName	.	.	.	Method	Injection
Shantel20181004	20181004_01_1	.	.	.	SV_PHENYL150X3MM3UM_ABA_GA_SIM4_Shantel.M	3uL

Sample Sequence

Tube	pos	reps	sample
ACN	91	1	MeCN
1	1	1	1) 1007 Cal_6d_imb

Tube	pos	reps	sample
2	2	1	2) 1008 Cal_6d_imb
3	3	1	3) 1009 Cal_6d_imb

Tube	pos	reps	sample
4	4	1	4) 1010 Cal_6d_imb
5	5	1	5) 1011 Cal_6d_imb
6	6	1	6) 1012 Cal_6d_dry
7	7	1	7)1013 Cal_6d_dry
8	8	1	8)1014 Cal_6d_dry
9	9	1	9)1015 Cal_6d_dry
10	10	1	10) 1016 Cal_6d_dry
11	11	1	11) 1017 Cay_6d_imb
12	12	1	12) 1018 Cay_6d_imb
13	13	1	13) 1019 Cay_6d_imb
14	14	1	14) 1020 Cay_6d_imb
15	15	1	15) 1021 Cay_6d_imb
16	16	1	16) 1022 Cay_6d_dry
17	17	1	17)1023 Cay_6d_dry
18	18	1	18) 1024 Cay_6d_dry
19	19	1	19) 1025 Cay_6d_dry
20	20	1	20) 1026 Cay_6d_dry
21	21	1	21) 1027 Cal-l_590-1_6d_imb
22	22	1	22) 1028 Cal-l_590-1_6d_imb
23	23	1	23)1029 Cal-l_590-1_6d_imb
24	24	1	24)1030 Cal-l_590-1_6d_imb
25	25	1	25)1031 Cal-l_590-1_6d_imb
26	26	1	26) 1042 DIST_6d_imb
27	27	1	27) 1043 DIST_6d_imb
28	28	1	28) 1044 DIST_6d_imb
29	29	1	29) 1045 DIST_6d_imb
30	30	1	30) 1046 DIST_6d_imb
31	31	1	31) 1047 DIST_6d_dry
32	32	1	32) 1048 DIST_6d_dry
33	33	1	33)1049 DIST_6d_dry
34	34	1	34) 1050 DIST_6d_dry
35	35	1	35) 1051 DIST_6d_dry
36	36	1	36) 1052 PROX_6d_imb
37	37	1	37) 1053 PROX_6d_imb
38	38	1	38)1054 PROX_6d_imb
39	39	1	39)1055 PROX_6d_imb
40	40	1	40) 1056 PROX_6d_imb
ACN	91??	2	MeCN

General Notes Run just the 1st sample first with what we think are what the retention times are, and we can fix it if necessary. And then run all the rest.

Our hormone profile standards look like this:

However, real samples will not be as crisp or clear. But running known endogenous amounts of each hormone allows us to know where each peak should be in reference of both the internal standard and the retention time.

Example of Sample :

Set 2 Week 2¶

Run Pameters

Folder	FileName	.	.	.	Method	Injection
Shantel20181010	20181010_01_1	.	.	.	SV_PHENYL150X3MM3UM_ABA_GA_SIM4_Shantel.M	3uL

Sample Sequence

Tube	pos	reps	sample
ACN	91	2	MeCN
1	1	1	1) 1057 PROX_6d_dry
2	2	1	2) 1058 PROX_6d_dry
3	3	1	3) 1059 PROX_6d_dry
4	4	1	4) 1060 PROX_6d_dry
ACN	91	2	MeCN
5	5	1	5) 1061 PROX_6d_dry
6	6	1	6) 1062 DIST_268-2_6d_imb
7	7	1	7) 1063 DIST_268-2_6d_imb
8	8	1	8) 1064 DIST_268-2_6d_imb
ACN	92	2	MeCN
9	9	1	9) 1065 DIST_268-2_6d_imb
10	10	1	10) 1067 PROX_472-1_6d_imb
11	11	1	11) 1068 PROX_472-1_6d_imb
12	12	1	12) 1069 PROX_472-1_6d_imb
ACN	92	2	MeCN
13	13	1	13)1070 PROX_472-1_6d_imb
14	14	1	14) 1071 PROX_472-1_6d_imb
15	15	1	15) 1072 Cal_0d_imb
16	16	1	16) 1073 Cal_0d_imb
ACN	93	2	MeCN
17	17	1	17) 1074 Cal_0d_imb
18	18	1	18) 1077 Cay_0d_imb
19	19	1	19) 1078 Cay_0d_imb
20	20	1	20) 1079 Cay_0d_imb
ACN	93	2	MeCN

Tube	pos	reps	sample
ACN	91	6	MeCN

Tube	pos	reps	sample
21	21	1	21) 1082 Cal-l_590-1_0d_imb
22	22	1	22) 1083 Cal-l_590-1_0d_imb
23	23	1	23) 1084 Cal-l_590-1_0d_imb
24	24	1	24) 1087 DIST_0d_imb
ACN	94	2	MeCN
25	25	1	25) 1088 DIST_0d_imb
26	26	1	26) 1089 DIST_0d_imb
27	27	1	27) 1090 DIST_0d_imb
28	28	1	28) 1091 DIST_0d_imb
ACN	94	2	MeCN
29	29	1	29) 1092 PROX_0d_imb
30	30	1	30) 1093 PROX_0d_imb
31	31	1	31) 1094 PROX_0d_imb
32	32	1	32) 1095 PROX_0d_imb
ACN	95	2	MeCN
33	33	1	33) 1096 PROX_0d_imb
34	34	1	34) 1097 DIST_268-2_0d_imb
35	35	1	35) 1098 DIST_268-2_0d_imb
36	36	1	36) 1099 DIST_268-2_0d_imb
ACN	95	2	MeCN
37	37	1	37) 1102 PROX_472-1_0d_imb
38	38	1	38) 1103 PROX_472-1_0d_imb
39	39	1	39) 1107 Cal_21d_imb
40	40	1	40) 1108 Cal_21d_imb
ACN	95	2	MeCN

General Notes
2018.10.11 - There was an error when I arrived in the morning. At 2:33:20am, Run 32 (MeCN Vial 93) had an error.
The LC (Sampler, Quant. Pump, and Column Comp.) all have a red error bar and the MS is green idle.
The screenshot log file is located here which contains the images of all of the errors reported.
G1316A:DEACN22825 - Error for which google lead me to the Agilent site describing that this is a G1316A/B Thermostatted Column

Repair Leak: 1) Turning the pump on at a lower 500mL/min, 2) while on, turning the knob to waste. 3) unscrew that the columns green screw end and 4) reseat it (just put it back) don’t overtighten 5) Turn knob back to column 6) check if still leaking

After a few attempts, Mel Oliver and I got the column to stop leaking worked, and the pump kept pressure and the column reached temp.

A	B	C

leak in green screw	Unscrewed	No Leak

Then run 6 washes MeCN. This will clean the system of any air bubbles from the leak.

To run the 6 washes, create the sequence, but do not save the sequence.

Then increase the number of MeCN washes between samples.

Set 3 Week 3¶

Run Pameters

Folder	FileName	.	.	.	Method	Injection
Sven20181004	20181004_01_1	.	.	.	SV_PHENYL150X3MM3UM_ABA_GA_SIM4_Shantel.M	3uL

Sample Sequence

Tube	pos	reps	sample
ACN	91	3	MeCN
1	1	1	1) 1109 Cal_21d_imb
2	2	1	2) 1110 Cal_21d_imb
3	3	1	3) 1111 Cal_21d_imb
4	4	1	4) 1112 Cal_21d_dry
ACN	91	3	MeCN
5	5	1	5) 1113 Cal_21d_dry
6	6	1	6) 1114 Cal_21d_dry
7	7	1	7) 1115 Cal_21d_dry
8	8	1	8) 1116 Cal_21d_dry
ACN	92	3	MeCN
9	9	1	9) 1117 Cay_21d_imb
10	10	1	10) 1118 Cay_21d_imb
11	11	1	11) 1119 Cay_21d_imb
12	12	1	12) 1122 Cay_21d_dry
ACN	92	3	MeCN
13	13	1	13) 1123 Cay_21d_dry
14	14	1	14) 1124 Cay_21d_dry
15	15	1	15) 1125 DIST_21d_imb
16	16	1	16) 1126 DIST_21d_imb
ACN	93	3	MeCN
17	17	1	17) 1127 DIST_21d_imb
18	18	1	18) 1128 DIST_21d_imb
19	19	1	19) 1129 DIST_21d_imb
20	20	1	20) 1130 DIST_21d_dry
ACN	93	3	MeCN
21	21	1	21) 1131 DIST_21d_dry
22	22	1	22) 1132 DIST_21d_dry
23	23	1	23) 1133 DIST_21d_dry
24	24	1	24) 1134 DIST_21d_dry
ACN	94	3	MeCN
25	25	1	25) 1135 PROX_21d_imb
26	26	1	26) 1136 PROX_21d_imb
27	27	1	27) 1137 PROX_21d_imb
28	28	1	28) 1138 PROX_21d_imb
ACN	94	3	MeCN
29	29	1	29) 1139 PROX_21d_imb
30	30	1	30) 1140 PROX_21d_dry

Tube	pos	reps	sample
ACN	91	5	MeCN
31	31	1	31) 1141 PROX_21d_dry
32	32	1	32) 1142 PROX_21d_dry
ACN	95	3	MeCN
33	33	1	33) 1143 PROX_21d_dry
34	34	1	34) 1144 PROX_21d_dry
ACN	95	4	MeCN

General Notes 2018.10.18 - There was an error after lunch on the 31st samples. At 12:42:28am, Run 63 (Vial 31, sample 1141) had an error.
The LC (Sampler, Quant. Pump, and Column Comp.) all have a red error bar and the MS is green idle.
The screenshot log file is located here which contains the images of all of the errors reported.
Vial 31 does have a puncture in the top, so 3uL was taken from the sample.

G1316A:DEACN2285 - Error
Instrument Error - Method/Sequence Stopped
G1316A:DEACN2285 - Leak Detected

The G1316A/B Thermostatted Column (green ended column as imaged above) is leaking again.
Repair Leak: 1) Turning the pump on at a lower 500mL/min, 2) while on, turning the knob to waste. 3) unscrew that the columns green screw end and 4) reseat it (just put it back) don’t overtighten 5) Turn knob back to column 6) check if still leaking

TOC

Troubleshoot LCMS Errors ¶

Always periodically check the side of the Nitrogen generator gages (on the side) and that the 2 lights are not blinking.

Look at the LCMS Windows to indicate which component has the errors.
Go to the run queue to see where the run stopped
Go to View Logbook can give some hints Current logbook (top is the most recent log)
View Logbook Sequence logbook to see any more info. sccroll down to when the instrument started the last sample, and view logs above that point.
Google the error (This may be helpful, but also confusing, but its a start)
Turn off MS Pump Yes
Turn off autosample physical button off, wait a few seconds, turn back on. The robotic arm will move around to calibrate itself.
Close the G6100 completely Yes Yes Yes, and re-open. (It may ask you to download the settings Upload from the instrument or to the instrument (we want FROM))
Turn the pump on 0.5 ml/min watch the pressure. If it suddently drops low (Or build up pressure really high). You can also visual look at the columns for leaks.
Try turning on the MS after it gets up to temperature .
If we have to shut the MS down, we need to do it very quickly, because there is gas in there to protect the MS, and it has pumps that keep it vacuumed, when we shut it down the pumps stop. There is a proper way to shut it down in the software (for long periods of shutdown). But we want to stop and restart it before the vacuum loses much.
First turn off the column. Double check MS is off. Shutdown software. YES YES NO to saving the current method changes. Turn off the LC first, then the MS. Turn on the MS then the LC.
If the MS still shows an error, we need to continue to fix the problem.

Common Errors:

- Take column cover off and see if its leaking. Overnight may have dried out, but it could have leaked. If it is leaking it will automatically shutdown. If this is the problem we can tighten the column and receed it. ¶

2018.10.05 Error (red) is in the MS window and ExShtDwn
       Error occured in sample #4, so inject a second time when we re-run
       Current Logbook: Instrument error refer to logbook occured at 8:10pm
       Sequence Logbook: APG RCI data overwrite error occured. timeout waiting for APG RCI.
       It might be a connection error with the software.
       Its possible the autosampler had the error, so we will turn it off and on.
We arent able to turn the MS window on and off. We restarted the machine, and there was no error and we were able to turn the MS on, and turn green.
       Bring the pump to 0.5mL/min, hit on, watch column for leaks.
If it works now, the arm probably had a movement error and based on Svens previous experience, this wont happen for a while from now. The sample vials have sample, and some condensation, for just this short amount of time, we will not adjust.
       If you are worried, put them in the speedvac to dry them down and re-dilute in 7uL (if the LSMC didnt sample, if it did, re-dilute 4uL).
Now that there are no visual errors, we set the run, and it appears to work.

LCMS Machine Run & Configuration ¶

The basis of the LC : sample/hormonw will flow based on both the chemistry in the column AND the size.
And the change of ratios of the solvents, you can push/pull out as slow or fast. Because if you have a low % solv B, things come out slower, but close peaks together seperate (more resolution of peaks close). But if you have a high % solvent B, things will come out faster. The same things with the disposal columns, but we arent keeping all, getting rid of things, this time we what to spread the hormones out, so when it gets shot out to the mass spec, it reads the masses.

1. LCMS Chemicals
First, Do we have enough solvents to run the LCMS Use HPLC graduate cylinder (in the hood). Rinse the cylinder with HPLC grade water before use When switching jars while on, turn the tube knob to waste

.	water + 0.1% Acetic Acid	Acetonitrile + 0.1% Acetic Acid	.	.
total	1700mL	1700mL	.	.
Acetic Acid	1.7mL	1.7mL	.	.
H2O/MeCN	Fill to total	Fill to total	.	.
.	.	Must mix very well or it will effect your results	.	.

Turn knob back to column.
MOST IMPORTANT THING TO REMEMBER WHEN CHANGING SOLVENTS When/if you change the solvents, you need to tel lthe computer how much is in the bottles. The machine will stop the run before it gets to 0mL.

Quant. Pump window click on the bottle letter. Ex: A change to 2.0 liters (full) with a max of 2.0 liters.
When you click, you can change all of the final volumes in the pop-up window.
Click Ok. Then the green liquid levels in the graphic will change.

A	B	C	D

**2. Turn on the pump 30min - 1hr before starting.**

Start with turning on the machine with a lower pressure (0.5) On
Once the temperature has reach around 60C, then change the pressure back to 1.0 (mL/min)

3. G6100 (online)
Sampler references black needle that will go from tube to tube. Idle box green (on)
Quant. Pump represents the A, B, C, D large chemicals. The clear tubes have letter labels. Only ever replace these one at a time, so there is no chance at switching tubes in the wrong liquid. Stanby box (grey offline)
Column Comp: represents the colume, and there are two shown on the screen because you can have 2 seperate columns and adjust the temp. Displays temp. Not ready (yellow) Watch the pressure to make sure its not too low (leak) too high at red bar (400mL/min) If it gets close to 500 it can handle it, but that might be a sign there is a clog or something thats not too normal. Too much pressure you can develope a leak, or cause issue with other tubes. MS: is the quadrapole shown for the mass spec (6120 Quadrapole MS)
Every time you run the LCMS - Method File icon to Load Method

SV_PHENYL150X3MM3UM_ABA_GA_SIM3.M (sven_column type_hormones_methodversionnumber)
scan will look at anything with all masses (so anything will show up, more noise that I dont want).
sim allows us to specify the MW of ABA, and then, MS, only scan at that MW. So you can do this on all the hormones you want to look at.
Signal 1 plot is the sample endogenous form, signal 2 is the IS form.

Change Method because we are exluding GA19 Method save as name the method in the folder c:\chem32\1\methods Edit Entire Method Leave all checked OK desciption of method you can edit not important OK ALS OK
We wont change the Solv A and B ratios, Keep the fow rate at 1.000 mL/min screenshot file of all of the settings we have the LCMS at. OK OK
Temperature fluctuations can effect peaks, so he keeps a LEFT high temp at 60C. And left and right are combined, so they both are the same. Set Up MSD Signal :Setting up the masses and the retention times we want the MS to start looking for our specific mass.

general tune file: atunes.tun ALWAYS put it on this one, no matter what anyone else tells you. It points to the most recent tune file.
Mode SIM keep that mode
Polarity, ABA/GA are negative, if you dont know run one with negative and then again with positive to see where your peaks show best.
GA19 Group4 Select cut both lines
Change Group 6 from 5.6 to 5.5 And do the same for the IS GA19 samples OK OK
The retention times for the Internal Standard or "endogenous" standards are differeent than when you run the samples.

Sample Purity: looking to M- no adduct (mass) and M-H (M - 1) in the negative tab since we are looking OK OK OK
Specify Report: G6100 Calssic Reporting check Screenuncheck File and the Quantitation setting is set to certian numbers based on the injection amount we did (24 and 48). And now everytime it runs, it should pop up on the screen. OK OK OK OK OK OK
Method Save Method MUST BE DONE now, or it will not run with this new updated method, it will run with the old method. Comment if you would like OK
Press giant ON button, hear a sound, thats the pump working. MS is still offline, when we actually start a run, the MS is online. and the temperatures are coming up to temperature.
Turn ON the MS Eveything should turn green and be ready. Once everything is ready, The top left tab will be green ready

General LCMS Notes B will go up during the run, but A will constantly output the same volume, so solvent A will run out quicker. Documents Shantel screenshot_logs
Lable files date_columnname_hormone HP Computer has no internet

Problems Step recorder
On windows 7 -Start -Search -PSR (Problems Step recorder) it will record screenshots evertime you click. -Start Recording - Stop recording - Save as Zip file. .mht file, click in file, opens in web browser. Settings - recent screenscatures to store, change to 100. You can copy this file onto a drive, put on laptop, and send to sven if need to trouble shoot

TOC

Running Dilution Curve ¶

What we are diluting is the endogenoud hormone (end) and we will keep the Internal standard (Is) will remain at the same concentration.

Calculate your dilution sample ng/ volume

A	B

Add to viles, spin down for 30min at 43C.
While that is spinning, update table (below) and add the sample sequence to the software/computer

Run Pameters

Folder	FileName	.	.	.	Method	Injection
Sven20181003	20181003_01_1	.	.	.	SV_PHENYL150X3MM3UM_ABA_GA_SIM4_Shantel.M	3uL

Sample Sequence

Tube	pos	reps	sample
ACN	91	1	MeCN
43	51	3	Is:end 8ng:2ng
42	52	3	Is:end 8ng:4ng
41	53	3	Is:end 8ng:8ng
ACN	91	2	MeCN

Notes:

Updating Software Sequence Info

Sequence Sequence Parameters Subdirectory: Sven20181003 Click Prefix cell (will ask to create, yes). Prefix should be the file name 20181003_01_ and the counter will start with 001
Check post seq command: STANDBY For a single run. If I wanted to run multiple runs, and whaat to run right after one another. You can change WHILE your sequence is running. Uncheck it if I dont want the LCMS to go to standby. OK
Sequence Sequence Table Click a cell and you can edit the cell. Input the pos into vial column. Highlight a row to cut it out. Insert to add more rows. Edit Sample Name, INjection, and Method Name needs to be my prefered method (all samples HAVE TO BE the same method).
Once the dilution samples are dried down, re-suspend to 12uL in 1% AcOH in MeCN.
Place tray in correct position according to my table.
Double Check Parameters, and Table. Method, Save Method.
To start the run: Run Control Rune Sequence a. If you ever need it to stop, you can press Pause Sequence then once it has paused after the current sample, THEN you can pressStop Run/Inj... and it will stop at the end of this sample (or the one following) but never in the middle of a sample.

TOC

Wheat Phenotype ¶

Purpose: To characterize the percent germination across multiple days of the different genotypes used in the Hormone and RNA experiments.

Hypothesis:

Line Selection

2018.09.13

Thawed 0d and 6d AR phenotype samples. Imbibed on 5mM MES wrapped in foil at room temp.

TOC

Wheat Hormone Tissue Harvest ¶

For each rep/Line Count
Imbibed_Hormone: 42 seeds | Imbibed_RNA: 21 seeds
Dry_Hormone: 47 seeds | Dry_RNA: 21 seeds

All 5 reps are sterilized and plated at the same time.
Hormone: 55 minutes to dissect all 5 reps embryos
RNA: 25 minutes to dissect all 5 reps embryos
Embryo: average 7 second time between first embryo incision and the sample put into liqN
HormoneDRY: 60 minutes to dissect all 5 reps embryos
RNADRY: 25 min utes to dissect all 5 reps embryos

Detailed experimental timeline was also recorded in my google calendar for Sept-Oct 2018. To view you need to sign into google.

Details of the tissue samples are in the Sample Details file. This includes the
-Info sheet with the header definitions
-Tissue_Data sheet that includes harvest, time points, etc of each individual sample
-Tiss_Summary sheet that contains any means or calculations needed to make decisions on how many embryos per sample should be done. -Extract_table and Peak_Data sheets include the information recorded for the hormone extraction. See Tissue Harvest Detailed Notes Week 1, Week2, Week3, LCMS Run Set Info for details on the experiment and General Lab Notebook TOC: Hormone Extraction for details on the peak calling analysis.

Experiment Notes:
Wed 2018.09.05

Thawed, counted, imbibed, and harvested all 6dAR Caldonia samples and started after-ripening remaining seed for 21d AR time point (700 seeds).
On average, it takes 15 min to harvest 40 imbibed seeds and about 10 min to harvest 20 imbibed seeds
It took 2 hours to harvest all of the dry seed samples (330 seeds). So around 41 seeds per 15 min.

Thu 2018.09.06

Thawed, counted, imbibed, and harvested all 6dAR IMBIBED Cayuga samples and started after-ripening remaining seed for 21d AR time point (414 seeds).
Thawed, counted and re-froze all 6dAR DRY Cayuga samples to be harvested on a less busy day.
Cayuga 21d AR will need to be reduced in the number of reps for dry seed (if not omitted).
Thawed, counted, imbibed, and harvested all 6dAR Calenoida-like samples (Imbibed samples only).
I realized, that this Cal-like time point should also mimic the QTL only samples, and I need to bulk these samples in the same manner as the QTL only samples. Therefore, I re-froze the remaining seed (543 seeds) at 6.5d AR to be bulked with the other 2 Cal-like BC1F8 families.
Its takes about 3 hours to stagger plating of all two genotypes samples and 3 hours to harvest without over imbibing (see definition below).
On average, it took 11 min to harvest 40 imbibed seeds and 5 min to harvest 20 imbibed seeds
Also there is an average 7 second time between first embryo incision and the sample put into liqN.

Fri 2018.09.07

Bulking the BC1F8 families involve taking 1/3 the amount of total needed for the whole (6d and 21d imbibed and dry) experiment from
Also only the Caledonia-like Family: 590-1 Plot#:2009 had enough extra seed plated/harvested YESTERDAY to use for my bulking method control.
I made a mistake, and bullked in Proximal QTL sample Family:459-1 Plot#: 2368 into the Caledonia-like bulking for 6d and 21d! It should have been Family:209-2 Plot#: 2338. There is no fixing this mistake, I now have contaminated samples for the caledonia-like control with a Proximal QTL mixed in. I have no extra seed to redo anythings Except for 0d AR. That will be the only time I can compare to the control.

Tues 2018.09.11

Fresh MES and sterilization solution made for plating assays today.
Imbibed and harvested embryo tissue from the DISTAL QTL Only bulked seed from the 237-3, 255-2, 268-2 families

Thu 2018.09.13

Imbibed and harvested embryo tissue from the PROXIMAL QTL Only bulked seed from the 282-3, 472-1, 501-1 families

Fri 2018.09.14

6dAR DRY Cayuga samples thawed and harvested
6dAR DRY Distal QTL only samples thawed and harvested

6dAR DRY Proximal QTL only samples thawed and harvested

[TOC](#TOC)

Instructions for James¶

Remove from Lyophilizer
1) Remove 2 boxes of 5ml tubes from the lyophilizer
2) Carefully remove the parafilm

a. some material may have stuck to the parafilm, so just remove the parafilm slowly so that no sample debris falls into another tube

3) Place the designated cap onto the corresponding tube.

a. Box 1 label goes with the box 1 tops, and same for box 2. Box 1 has clearly labeled A1, one the box top and box, but Box2 two has a pink tape corner that matches up Box 2 box with box 2 top.
b. The final weight depends on the precise tube + cap. So each tube has a number that goes with the same number on the cap c. I placed the caps in the same order within the box tops
d. Some caps may be dirty and I remember being excess, I wouldn’t be too worries, but if see one that has too much un-lyophilized sample remaining on the tube, you can wipe with kimwipes if you need.

Final Weight
Weigh the 5ml tube + sample material + cap
1) Use the small scale
2) Place the small grey tube holder sponge onto the scale and tare that.

a) Tube holder is on the shelf on top of the parafilm box next to the brush.
b) I use this to keep the tube very consistently still on the scale to get a precise weight.

3) I have a “empty balance tube” that I use to make sure the scale is measuring consistently. Weigh that tube (currently set in the small grey tube holder) a couple times and record the weight of the balance tube in the google sheet in the “Info “ sheet.

a) https://docs.google.com/spreadsheets/d/1x9ebbxZ2tzYSNwGcHoq-qzIoFzxiQ0qNi48rBD-R2-g/edit#gid=0
b) Ive noticed the tube size is decreasing over time, which is odd, and I think MAYBE its because the scale has been moved a few times since my first round of weights. But I have been making sure to make sure the scale is level everytime I weigh, and I place the tube in the grey holder exactly in the middle of the scale everytime to stay consistent.
c) But, the very least, I would like to know what the “empty balance” tube weighs the day these weights are taken.

4) Weigh the samples and enter in the all 4 decimal digits into the google sheet “Data” sheet in the column “weight_sample” for the correct sample number in the “tube” column.

a) I need to keep track of the mg difference, so all 4 digits are important.
b) https://docs.google.com/spreadsheets/d/1x9ebbxZ2tzYSNwGcHoq-qzIoFzxiQ0qNi48rBD-R2-g/edit#gid=0

Prep for Shipping
Parafilm tubes and place in white boxes to ship.
They fit pretty snug in the boxes. I left two boxes near my bench area. Since you will ship these overnight during a weekday, Im not too too worried about putting the boxes in another bag to keep it dry, a good parafilm should suffice for the journey.

TOC

Key Tips

Keep the spacing of the seeds the same density in the hormone (46 seeds) plates and the RNA plates (20 seeds)
Every morning, double check the liq N tank BEFORE thawing or sterilizing any seeds. If there is no liqN late at night when I harvest tissue, there is a major loss in crucial seed.
Stagger plating assays so that the time the seeds are plated (out of the tube and onto the plate, even prior to organizing and parafilming) + 8 hrs is as close to when the seeds are harvested. Remember it takes about 75 minutes to dissect all 5 reps embryos for the hormone samples and about 50 minutes to dissect all 5 reps embryos for the RNA samples. Therefore you cant plate every 30 minutes in the morning, or else you wont be able to dissect emryos fast enough to hit every 8hr time point, meaning you will over imbibe 8++ hours.

To prove the bulking for hormone of the QTL samples is valid, I would like

5 reps of each line imbibed for 8hrs at 6dAR to also show that the bulked 6dAR imbibed samples showed similiar results as the lines separately.
The same goes for the RNA extraction: 5 reps of each line imbibed for 8hrs at 6dAR

Sample Condition Cheatsheet for each genotype

Sample_Num	AR_Day	Imb	Exp	Rep	No Seeds	TOTAL
1	6	8	Hormone	1	40
2	6	8	Hormone	2	40
3	6	8	Hormone	3	40
4	6	8	Hormone	4	40
5	6	8	Hormone	5	40
6	6	0	Hormone	1	46
7	6	0	Hormone	2	46
8	6	0	Hormone	3	46
9	6	0	Hormone	4	46
10	6	0	Hormone	5	46
11	6	8	RNA	1	21
12	6	8	RNA	2	21
13	6	8	RNA	3	21
14	6	8	RNA	4	21
15	6	8	RNA	5	21
16	6	0	RNA	1	21
17	6	0	RNA	2	21
18	6	0	RNA	3	21
19	6	0	RNA	4	21
20	6	0	RNA	5	21
21	6	-	Pheno	-	31	671

22	21	8	Hormone	1	40
23	21	8	Hormone	2	40
24	21	8	Hormone	3	40
25	21	8	Hormone	4	40
26	21	8	Hormone	5	40
27	21	0	Hormone	1	46
28	21	0	Hormone	2	46
29	21	0	Hormone	3	46
30	21	0	Hormone	4	46
31	21	0	Hormone	5	46
32	21	8	RNA	1	21
33	21	8	RNA	2	21
34	21	8	RNA	3	21
35	21	8	RNA	4	21
36	21	8	RNA	5	21
37	21	0	RNA	1	21
38	21	0	RNA	2	21
39	21	0	RNA	3	21
40	21	0	RNA	4	21
41	21	0	RNA	5	21
42	21	-	Pheno	-	31	671

43	0	8	Hormone	1	40
44	0	8	Hormone	2	40
45	0	8	Hormone	3	40
46	0	8	Hormone	4	-
47	0	8	Hormone	5	-
48	0	8	RNA	1	21
49	0	8	RNA	2	21
50	0	8	RNA	3	21
51	0	8	RNA	4	-
52	0	8	RNA	5	-
53	0	-	Pheno	-	31	214

TOC

Dry Weight Per Embryo Experiment ¶

PURPOSE: Define the number of embryos needed per sample to have the minimum amount of 50mg dry weight for imbibed seeds and 100mg of dry weight for dry seeds.

HYPOTHESIS:
Imbibed embryos for 8 hrs will weigh more than dry embryos. 30 embryos will be enough for imbibed seeds, but the 30 embryos of the dry samples will not be enough for the minimum weight and will need more embryos.

EXPERIMENTAL DESIGN:
Samples: 6d AR Caledonia
8hr imbibed: 10 | 20 | 30 embryos 2018.08.23 Sterilized: 9am Plated: 9:30am Dissected: 5pm
0hr imb/dry: 10 | 20 | 30^ embryos 2018.08.23 Dissected: 5:30pm ^this sample is 26d AR

The germination assay protocol was followed except the imbibed samples were plated on 6mL of autoclaved water instead of buffered 5mM MES. This is because the MES was cooled in the fridge and I could not get it to open (oops), and since this is just a preliminary experiment, I used water instead.

The emrbyo dissection was done under regular lab lighting. During the hormone experiment sample prep, dissection needs to be done under green safe light to prevent any photosynthetic signals. Since we are only measuring hormone content instead of RNA expression, its not as necessary to dissect under the greenlight. However, an RNA expression study is likely to take place, and I would prefer the exact same conditions done on all samples to potentially compare later.

Caledonia is very after-ripened by 6 days, and about 3 seeds out of the 60 imbibed seeds showed visible germination. For all future experiments, always plate 2 extra seeds in addition to the determined number of seeds per condition, just in case seeds are germinated, then you can dissect the extra seeds and not the visibly germinated seeds.

This experiment and all subsequent experiments will only include samples of un-germinated seeds. A comparison of initial seedling/radicals to non-dormant ungerminated seeds is not desired.

Measuring dry weight for hormone extraction protocol was followed.

All of the tube information is found in this google worksheet.

RESULTS:
Dataset analysis in googlesheets file under Summary sheet
Data

geno	ar_days	imb_hrs	sterilized	order_ground	no_embryos	dry_weight_mg
Caledonia	6	0	N	5	10	6.9
Caledonia	6	0	N	3	20	25.7
Caledonia	26	0	N	2	30	39.5
Caledonia	6	8	Y	6	10	10.1
Caledonia	6	8	Y	1	20	33.9
Caledonia	6	8	Y	4	30	19.9*

Analysis

imb_hrs	rep1_10	rep2_10	rep3_10	avg 10 embryo
0	6.90	12.85	13.17	10.97
6	10.1	16.95	6.63	11.23

CONCLUSION:

Sample type	Based on 20E sample	Recommendation
	Number	of embryos needed
dry	78	90
imbibed	29	40

It looks like 90 embryos per dry sample and 40 embryos per imbibed sample will give both conditions enough matter to reach our preference weight of 100mg and 50mg dry weight, respectively.

I don't think I'm limited on seed based on my estimates for this hormone experiment, but I would like to keep a lot of seed from this experiment set to extract RNA with. If we are going to increase anything, we should only increase the dry embryo sample number.

SNelson: It should be ok as it is, but GA is generally at low abundance in dry seeds, so that is the place to increase if you find you have seed to spare. Also, I think 100mg of embryos will probably be better than 100mg of seeds (embryos and everything else) because the dead starchy area may not have a lot of hormone,

A googlesheet has the tentative experimental designs. I can update it and change it in case it's necessary to increase the embryo number. Changing the embryo number will change the early time points. But no matter what, I know I'm limited in the total number of samples (280-300) I can run. I have 4 scenarios laid out in the experimental design file . Optimizing the number of samples I can run while I'm at University of Missouri and knowing how many seeds I have at the 0 d AR time point, I think I can run 9 different lines instead of my original idea of 2 in the proposal. Notice though the caveat that I can not run dry seeds at 0d AR. No matter what scenario I pick or how many lines I compare. All I will be able to compare for the dry conditions is the 6 and 12 d AR time points and the lines.

Overall, this project will be mainly focused on comparing hormone levels and RNA expression of hormones/precursors of:

Genotypes a) Cayuga, b) Caledonia, c) three BC1 lines with only the distal QTL, d) three BC1 lines with only the proximal QTL, and e) a BC1 line that is Caldonia-like in genotype and phenotype.
After-ripening time points of 6d and 12d across all genotypes. 6 days has the large difference between Cayuga and Caledonia phenotype, and 12 days is when PHS tolerant Cayuga is losing more dormancy (so more near a comparison of both being after-ripened).
Dry seeds versus imbibed seeds at both 6d and 12d AR.
Early after-ripening stage 0d imbibed 6 and 12 hours will be a small bonus comparison.

Concerns: Thoughts on having 2 different embryo numbers for dry versus imbibed if we want to compare the two later? I would love the same number of embryos in the dry seed experiment the same number as the imbibed samples, to keep the experimental design balanced, but that seems like so much.

SNelson: My thought on having 2 different embryo numbers for dry vs imbibed and comparing them later? This is not a problem, it is best to do it this way. Even if you had unlimited seed, you would want to use more for dry than imbibed, since you would hit the maximum for imbibed seed earlier than for dry. It’s just the nature of doing these sorts of extractions/analysis. In the end, your result will essentially give you the mean of all embryos that went into one sample, so more embryos will increase the accuracy of your mean (you will observe less of the variance). So it would be better not to compare 1 embryo to 100 embryos, since you will observe more sample-to-sample variation when only sampling an individual seed, but with 40 embryos or 90 embryos your results should be getting quite close to the actual mean.

Thoughts on me just doing a dry and 8hr imbibed timepoint. You think its needed to do a dry, 0hr, and 8hr imbibed time points?

SNelson: Can you refresh me on what the 0h is? How different is that from dry? Do you have a cold treatment or anything between dry and 0h or is it just adding water and then sampling? If it is just adding water and sampling, then I would say you can cut that one. If you have enough seeds, though, you might move it back an hour or so to get the eariest transcribed genes after imbibition. We can discuss more based on your answer to above.

Be sure to have enough seed for the gene expression portion of the project.

CC Field Parental Germination Assay ¶

Purpose: To get a better understanding of Caygua's and Caledonia's germination response over and after-ripening time course.

Hypothesis: Based on the greenhouse germination assay, there should be a large difference between Cayuga and Caledonia within 2 weeks of ARing (14 days). Since the GH results in increased dormancy, I expect dormancy loss to occur earlier in the field material.

Line Selection
The NIFA grant (Objective 2) included comparing 2 lines, but I've upped it to 3 lines:

one with the proximal 2B QTL genotype, with consistent PHS tolerance
one with the distal 2B QTL genotype, with consistent PHS tolerance
one without either QTL genotype (NR-Cal) with consistent PHS susceptibility

The PHS scores I'm looking at are data from 2009-2011, and I only selected lines that showed consistency (had low variation across years) .

I have 5 lines selected in each category. And based on our 2018 emergence data, they all appear to have a lot of plants/spikes in the field. I will harvest all 15 lines and double check their genotyping before moving forward with the sample prep.

I am going to harvest samples for analyzing both the 2B QTLs, because why not have the materials in case I want to look at both QTLs hormone profiles. I may later have to just pick one, since that was in our original budget, but I can decide that later if money is an issue.

Now I think that problems could arise if I select lines with and without the 2B QTL region, and they have or do not have another dormancy QTL that Munkvold found on another chromosome. But I think I can sift through Munkvolds older data and see what genotype each line showed in that original analysis. OR I can harvest and combine 3-5 lines per category and pool them before the hormone experiment prep to represent only the 2B QTLs. And hopefully by pooling the samples, that will drown out 'other' dormancy QTLs.

Lines were harvested from Snyder 2018 instead of Helfer 2018, because Helfer had too much bird damage, even though Helfer had a better percentage of emergence

Experimental Design

I have 5 spikes at 0 days AR for all the lines I harvested for the Hormone Experiment. Then I have a larger amount of lines at 6 days AR. I harvested quiet a bit of Cayuga and Caledonia in addition to the lines for the Hormone Experiment.

So, is 6 days of after-ripening to late in the field material? Or is it a good intermediate time point.

In the end, I would like an early (dormant), mid (losing dormancy), and late (dormancy loss) AR times point for all the lines in the hormone experiment.

2018.08.03 Sterilized and plated 6d AR seeds on 5mM MES
2018.08.04 Sterilized and plated 7d AR seeds on 5mM MES
2018.08.05 Sterilized and plated 8d AR seeds on 5mM MES
2018.08.07 Sterilized and plated 10d AR seeds on 5mM MES

A	B

CC Greenhouse Parental Germination Assay ¶

Purpose: To get a better understanding of Caygua's and Caledonia's germination response over and after-ripening time course from greenhouse harvested material.

Hypothesis: We expect to see a large difference between Cayuga and Caledonia after so many day of after-ripening. The GH increases dormancy because of the environment during maturation, so we expect dormancy loss to occur earlier in the field material.

Experimental Design

      Lines: Cayuga | Caledonia
      ARing time points (days): 6 | 7 | 8 | 9| 10 | 13
      No. seed per condition: 20 seed
      Total seed per line: 120 seeds

So, is 6 days of after-ripening too early in the greenhouse material? Or is it a good starting time point.

Goal: In order to phenotype the BC1F8 seeds, I want the time point that gives the largest percent germination between the parents.

Results:
The raw data is found in the shared google drive file

A	B

TOC

Lab Protocols ¶

Extraction for Hormone Measurement (USDA-ARS MU 2017) ¶

Author: Sven C. Nelson, PhD | Comments: Shantel A. Martinez, PhD
This is the extraction of samples for ABA and GA (and GA precursor) measurement in Columbia, Missouri (USDA-ARS in Oliver Lab) using LCMS. Based on the protocol from RIKEN 2013, but with substitutions for tools that we do not have here. The 2016 version of this protocol used 15 mL tubes, but it has now been further modified to use 5 mL polypropylene round-bottomed tubes (Falcon, product: 352063) for most steps. This allows speed-vac steps to accommodate 40 tubes at once with our new rotor for 5 mL tubes. For larger samples, refer to 15 mL or 50 mL tube protocols. (Max 400 mg sample : 50 mL tubes.)

5 mL tubes used: 1

**START DAY 0**
1. Measure the weight of the freeze-dried samples in 5 mL tube. (desired: 50-100 mg DW)

a. Do this beforehand. Pre-weigh empty tubes before adding ground tissue and then weigh again after freeze drying. Subtract the first from the second to get the total dry weight. Be sure to use the sensitive scale and record all 4 digits after the decimal for your pre-weights.

5 mL tubes used: 0

**START DAY 1**
**↧ ON BENCH OR IN HOOD**
2. Add 2 mL of the 1st extraction solution to each samples

a. For all extraction solutions, use HPLC quality H2O. Don’t store extraction solution as it contains internal standards (ISs) which can degrade over time, make fresh.
b. 1st extraction solution: 80% Acetone containing 1% AcOH and IS-MIX solution
c. Add IS (GA and ABA internal standards) to extraction solution “master mix” so that each sample will get the desired final ng per sample. Record the per-sample ng of IS added.
d. How much IS-MIX to add? Decide based on amount of endogenous GA/ABA expected and peaks within the linear range of detection in test LCMS runs of ISs.
e. Example: If 7 μl final volume (per sample) and injecting 3 μl. If you desire 24 ng/injection, then there should be 56 ng/sample (24 × 7/3). However we are adding 2mL per samples, therefore 56/2 = 28 ng/1mL.

.

Chemical	Concntration	40mL	45mL	unit
Acetone	80%	32	36	mL
AcOH	1%	400	450	μL
Internal Standard Mix	24ng/inj & 48ng/inj	291.2	326.8	μL
H2O	-	7.6	8.55	mL

A	B

3. After mixing by vortex, incubate the tube for 6 hours (or ON) at 4ºC. (1st incubation)

5 mL tubes used: 1

**START DAY 2**
4. After 6 hours, remove caps, centrifuge samples at 4000 rpm (3399 g) for 10 min.

a. Large centrifuge, max 5100 rpm. Settings: S.51 and set PLA/BUC to BUC (buckets).
b. While spinning 1) label new tubes (tube set #2) 1-40 if you havent already done so, 2) make 2nd Extraction solution (see step 6), and 3) Turn on speedvac with vapor trap (it takes 90min to reach temperature; see step 10).

5. Collect the supernatant in labeled 5 mL tubes (save the supernatent tube set #2 until step 9).

a. Use a 1mL pipette with fresh tips very time.
b. The supernatent tube set #2 can be left on the bench (or in a drawer to keep IS from degrading in the dark) or at 4C if left for a longer period of time.

6. Add 2 mL of the 2nd extraction solution to the pellet (1st tube set) of each sample. with repeat pipettor, reusable tips undr the hood, 1mL x2.

a. 2nd extraction solution: 80% Acetone containing 1% AcOH. Make this with the units on the falcon tube).
b. Make 3x 50mL falcon tubes, the solution can sit over time. c. re-cap tubes for next step.

Chemical	Concntration	50mL	unit
Acetone	80%	40	mL
AcOH	1%	500	μL
H2O	-	fill to 50mL	mL

7. After mixing by vortex, incubate the tubes for 10 min at 4ºC. (2nd incubation) Votex time: count to 10
8. Centrifuge samples without caps at 4000 rpm (3399 g) for 10 min.
9. Collect the supernatant (tube set #1) and add to the same 5 mL tube (tube set #2) from step 5 (discard pellet)

a. Use a 1mL pipette with fresh tips very time.

10. Evaporate extraction solution using speedvac in lab (new 40 × 5 mL tube rotor)

a. 43-45°C, vacuum level 20.0, heat time 4hrs, time required: ≈3 hours (it’s ok if ≈0.5 mL or less remaining) b. To turn speed vac on: switch speedvac switch on, vapor trap swtich on, pump switch on (if it didnt turn on with the vapor trap)
c. Change the rotor 0-12 that fits 40 5mL falcon tubes.
d. The vapor trap needs to reach the temperature -104C (takes ~90min)

LUNCH

**↧ IN HOOD**
11. Add 2 mL of Hexane to the dried down sample.

We are using the Hexane to pull out un-wanted junk

12. Add 2 mL of 50% Acetonitrile (MeCN) and vortex.

a. Solution will form two layers or can (but not necessary) be centrifuged at 8000 rpm for 5 min

13. (3×) Remove the upper layer (Hexane) and discard, add 2 mL Hexane.

a. 3× = do this a total of 3 times, check the first box and repeat two more times: ☐ ☐ ☐
b. Do not add Hexane on the 3rd time and move on to step 14
c. In layman's terms: 1) remove Hexane layer, discard, 2) add Hexane, vortex, remove Hexane layer, discard and 3) add Hexane, vortex, remove Hexane layer, discard. Proceed to Step 14

14. Evaporate extraction solution using speedvac (no heat, run overnight OR 43°C, ≈3-4 hr).

a. For the next day: Make PVP solution (50/50 of PVP and HPLC-grade water) if needed.

PREP FOR TOMORROW: Make 0.5M K2HPO4 soln (see step 15 notes); Label 2 sets of tubes; Make 1% AcOHaq

5 mL tubes used: 2

**START DAY 3**
**↧ ON BENCH**
15. Add 0.5 mL 0.5M K2HPO4 to the dried down sample, vortex, and allow to dissolve.

a. Add 4.355 g to total of 50 mL of water; MW = 174.18
b. While waiting for samples to dissolve, prepare and condition the PVP columns (step 16)

16. Conditioning the PVP column (PVP = Poly(vinylpolypyrrolidone), ex: Sigma prod. 77627)

a. Fill Varian Reservoir Frits 1 mL columns (Agilent 12131013) to 1/2 volume with PVP

i. To do this, add 1 mL of 50/50 mix of PVP and HPLC-grade water to column.
ii. Use a syringe with column adapter to gently push through some water to start the gravity-flow of liquid through the column
iii. Check pH of flow through with lithmus/pH paper, it is probably about pH 7
iv. Add ≈500μl each time of 0.5M K2HPO4 up to 2 mL (4 times) or until the pH gets to about pH8~9 (Check flow-through with litmus/pH paper) ☐ ☐ ☐ ☐

17. Collect flow-through: Once dissolved load the 0.5 mL sample in the PVP column, add another 0.5 mL 0.5M K2HPO4 to the sample tube and add this to the column. ☐ ☐

a. (optional) If sample is dirty, can first pass through and empty 3mL Varian Reservoir Frits column (0.5 mL × 2) to remove larger debris, then add that 1 mL to the PVP columns**
b. lift rack to see if liquid went through & tap to let last drop fall.
c. PVP/column can be discarded in regular trash

18. Collect flow-through: add 2 mL 0.5M K2HPO4 (4 × 500μl) to elute. ☐ ☐ ☐ ☐

a. Total volume of flow-through is 3 mL

LUNCH *suggested stopping point, but any break above works too. I tend to eat during step 18 while I wait*

**↧ IN HOOD**
Turn on speed vac + vapor trap (90min prior to step 24)
19. Add 6N HCl 100 - 300 μl to the sample until the pH is 2 - 3, check by litmus/pH paper.

a. add 100uL to all, spin uncapped, check multiple tube pH's. b. add 100uL to the #1 tube, check pH, add 100uL more if you need more to get 2-3 pH.
c. then add 200uL to all of the tubes (or 100uL if you only needed 100uL more instead of 200uL).
d. spin uncapped, check pH, recap, and set in the dark drawer, and procees to step 20

20. Conditioning the 3 mL HLB column (Oasis HLB 3cc Vac Cartridge, Waters WAT094226)

a. Add 3 mL of MeCN and discard flow-through
b. Then 3 mL of MeOH and discard flow-through
c. Then 3 mL of 1% AcOHaq and discard flow-through

Columns are snug in the column holder, dont need to push all the way down.
Constantly remove the waste solution into properly labeled Waste Container

Chemical	Conc.	Amount	unit
AcOH	1%	1	mL
LCMS H2O	-	99	mL

21. Discard flow-through: Load the extracts to HLB (ABA/GA is retained in column).
22. Discard flow-through: Add 2 mL of 1% AcOHaq twice (2× wash step). ☐ ☐

a. Near the end, you can use the syringe to push the column solution all the way through, to get that extra last bit out. However push slowly and steady.

23. Collect flow-through: Move columns above new 5 mL tubes, add 2 mL 80% MeCN containing 1% AcOH twice (2× elution step). ☐ ☐

4mL/sample * 40 samples = 160mL

Chemical	Concntration	160mL	unit
MeCH	80%	128	mL
AcOH	1%	1.6	mL
H2O	-	30.4	mL

24. Evaporate extraction solution using speedvac (43°C, run overnight OR 43°C, ≈4-5 hr). no liquid left

5 mL tubes used: 2

**START DAY 4**
**↧ IN HOOD**
25. Add 0.5mL MeOH to each of the dried down samples, pellet will dissolve easily, vortex briefly to mix.

*DO NOT ALOW COLUMNS TODAY TO DRY OUT*

26. Conditioning the 1 mL DEA columns (Bond Elut DEA cartridge, Agilent 14102016)

a. Add 1 mL MeOH and discard flow-through

27. Discard flow-through: Load the extracts into the DEA columns (ABA/GA is retained).
28. Discard flow-through: Add 1 mL MeOH three times (3× wash). ☐ ☐ ☐
29. Collect flow-through: Move columns above new 5 mL tubes: Add 1 mL MeOH containing 0.5% AcOH three times (3× elution step). ☐ ☐ ☐

Chemical	Concntration	150mL	unit
MeOH	-	150	mL
AcOH	0.5%	750	μL

30. Evaporate extraction solution using speedvac (43°C, ≈2 hr).

a. Place at 4°C if dry before lunch.

LUNCH

31. Add 0.5 mL of 50% Chloroform 50% ethylacetate containing 0.1% AcOH to the dried down samples, vortex briefly.
32. Conditioning 1 mL Sep-Pac Silica columns (Sep-Pac Silica 1cc, Waters WAT023595)

a. Add 3 × 1 mL of 50% Chloroform 50% ethylacetate 0.1% AcOH (discard) ☐ ☐ ☐

Chemical	Concntration	440mL	unit
Chloroform	50%	220	mL
Ethylacetate	50%	220	mL
AcOH	0.1%	440	μL

33. Collect flow-through: Load extracts to Silica and collect in pre-labeled 5 mL tubes.
34. Collect flow-through: Add 1 mL 50% Chloroform 50% ethylacetate containing 0.1% AcOH twice (2× elution step; total 2.5 mL in tube) ☐ ☐
35. Evaporate extraction solution using speedvac (43°C, ≈1 hr).

Can prep the LC/MS vials in for step 36

*A time you can store @ RT for 1-2hr OR @4C >2hrs*

36. Resuspend in 200μl MeOH, transfer to LC/MS vial insert, dry by speedvac (43°C, 30 m).

For steps 36 and 37, you can use a speedvac without a vapor trap.

37. Repeat step 36: Add 200μl MeOH, transfer from 5 mL tube to vial insert, and dry down.

a. Store in this form and resuspend for run.

38. For small injection sizes (eg 3μl per inj), resuspend in X μl of 99% MeCN 1% AcOH.
(X = inject vol × # of inj + extra; ex: 7μl; higher MeCN is needed for GA12/12-Ald peaks)
For larger injections (eg 20μl per inj), resuspend in X μl of 15% MeCN 1% AcOHaq.

PROCEED TO RUNNING SAMPLES ON LCMS

Chemical Locations
acetone under hood in other lab area
acoh in hood next to lcms
water aliqout in 50ml hplc grade tube, so its easier to use. water can be pipetted
6M HCL Acid cabinet Columns HLB 3mL/3cc Sven LCMS Column drawer K2HPO4 stock inorganics cupboard; 0.5M solution, above benc.
PVP Organics cupboard ~110um particle size
1% AcOHaq larger 1L brown bottle Bottles of LCMS water, are under desk area, large brown bottles of water (if I get low on it, tell kate and she will ordere more)
Internal-standard mix solution in the -20C Sven Box
ga's IS stock tip box 50ug/ml
+aba IS stock tip box 1mg/ml

TOC

Measuring Dry Weight for Hormone Extraction ¶

Step 1: Label each tube with an idenifing number.

My labeling system is just the numbers in the 1000's, and all of he tube information is found in this google worksheet.
For the hormone extaction protocol, 5 mL polypropylene round-bottomed tubes (Falcon, product: 352063) is needed for downstream speed vac rotor size.

Step 2: Place tube holder (Below Image A) on the small balance.
Step 3: Close the balance door. Wait 20 seconds for the tube holder to steady. Tare the tube holder.

From this point on, always be patient. The precise weight is more important than the time it takes to tare, wait, weigh, wait, etc.

Step 4: Add the empty tube plus cap into the designated tube holding position. Close the balance door. Wait until the balance read stops on one value for 15+ seconds.

Changing where a tube is measured from tube to tube will result in inconsistent values, increasing the measuring error. So keep the position of the tube exactly the same for each tube, and preferably in the exact middle of the balance.

Step 5: Record the empty weight value into the datasheet column "weight_empty". Record all 4 digits past zero. Repeat steps 3-5 for each tube.

The sample masses are key to determining the final concentration, so use the most sensitive scale that you have and record all 4 digits past the decimal. (Ex. 7.0035g - 6.0125g = 0.091g) This information is used in the final data analysis to determine ng/g dry weight of the hormone in your samples.

Step 6: Prep samples per experimental conditions
Step 7: Flash freeze samples in liquid N
Step 8: Grind in liquid N. Make sure tissue is well ground. Place in -80C for a minimum 2 hours.

For dry seed tissue, add a few drops of water into the liquid N with the seeds before grinding to help break up the tissue.

Step 9: Separate the lids and cover the tubes with parafilm that have poked holes from a tack.
Step 10: Lyophilize ground sample for 48hrs (Below Image B).
Step 11: Replace tube lids with original lids (Below Image C). Keep track of which lid goes with which tube since each lid has a slightly different mass
Step 12: Weigh the tube with lid now containing the lyophilized sample as in Steps 3-5 above. Record the sample weight value into the datasheet column "weight_sample". With the "weight_sample" minus the "weight_empty", we can get the actual mass of the lyophilized sample (column “dry_weight”).
Step 13: Parafilm the tubes tightly to prevent any sample from escaping and to prevent moisture entering during shipping.
Step 14: Store and ship the tubes wrapped in aluminum foil to prevent any hormone breakdown due to light. You can store and ship these samples at room temperature.

A	B	C

Lyophilizer ¶

Set-up/Run
Step 1. Check the drain. Pull the tubes plug and drain the excess water into a tub

If the lyophilizer is in heavy use, it will have a lot of built up moisture.

Step 2. Turn the machine on. On/Off switch is on the right side
Step 3. Turn on the refridgerator

There are three main buttons: Two of them are for cooling. Auto and Man. Use manual so that the vacuum doesnt automatically turn on once cooled.
Wait for the temperature to come down to -40C - -50C (only takes about 10 min)
Warning: the plastic dust cap will fly off, you didnt break anything. It just keeps dust out for long term storage.

Step 4. Add your samples into the chamber and close the chamber. Double check valves are closed. (Notch on top)
Step 5. Turn on vacuum
Step 6. In a few minutes, double check to see if te lyophilizer is pulling vacuum.

Note: It won't reach full green vacuum, because James has it set that way. The second to last light will blink.

Turn-off
Step 1. Break the vacuumm slowly by opening a valve
Step 2. Turn off the vacuum pump button

If you know your samples are finished, you may take them out now and proceed with shutting down lyophilizer.
If the samples are not fully dried, repeat steps 4-6 above for additional hours of freeze drying.

Step 3. Turn off the refridgerator button
Step 4. Turn unit off switch

Remove the chamber and let thaw.
Wait until fully thawed before drainin the excess water.

Germination Assay ¶

Seed Sterilization
Step 1. Count out number of seeds (dry) and place the seeds in 50ml conical tubes

a. One line per tube, 3 reps of 10 = 30 seeds per treatment
b. Treatments may include 2uM ABA, 5uM ABA, 5mM MES (No Hormone), or 10uM GA

Step2. Add sterilization solution 5-10ml above dry seed line (not rocket science). Tightly screw lid.
Step3. Shake to mix every 5 min for 15 min.
Step4. Use the vacuum with a tip to suck out the solution (It’s hard to get out all solution, but try to get as much as you can)
Step5. Add autoclaved dH2O 5-10ml above seed line (to dilute and wash out the sterilization solution)
Step6. Shake every few minutes for 7min
Step7. Vacuum off dH2O and repeat steps 5 & 6 twice (a total of 3 rinses)
Step8. Use the vacuum to suck off autoclaved dH2O

Plating seeds
Step1. Use sterile petri plates and sterile filter paper; insert one blue filter paper into each plate
Step2. Label the top of each plate according to solution concentration, type of seed being plated, weeks after-ripened, and date plated
Step3. Make hormone/treatment solutions and add 6ml into each plate

6 mL per plate | Calculate 6.25 mL x Number of Plates

Step4. Use small forceps to remove bubbles underneath filter paper (make sure the filter paper is flat on the bottom, may need to fold on the sides to fit)
Step5. Use small forceps to place each sterile seed onto the plate (embryo facing left, seed fold facing down)
Step6. Plate 3 sets of 10 onto one plate
Step7. Parafilm the top and bottom of the plate together in order to keep the moisture in
Step8. Wrap 3-4 plates together in foil to exclude light. (shiny side in; dull side out)

Germination Screen
Step1. Once plated, place in the 20-22°C incubator
Step2. Score every 24 hours for 5 days after plated and incubated
Step3. Day 1: mark on top of plate / for germinated seeds and X for 3-point seeds

a. Record both germinated seeds and 3-point seeds for each day

Step4. Day2: record newly developed germinated and 3-point seeds
Step5. Continue process on Day3, Day4, and Day5

Germination Index
(5 x gday1 + 4 x gday2… + 1 x gday5)/(5 x n)
Where gday1 is the number of seed that germinated on day 1
gday2 is the number of seeds that ADDITIONALLY germinated on day 2 and so on
n is the total number of seeds plated

Sterilization Solution: 10% Bleach + 0.01% SDS + ddH2O

Total Vol.	200mL	400mL	800mL
Bleach	20mL	40mL	80mL
2%SDS	1mL	2mL	4mL
ddH2O	179mL	358mL	716mL

TOC

DNA extraction ¶

James Tanaka | 2018.06.06

Harvest Tissue
Step 1: Place two metallic grinding beads in each sample well.
Step 2: At the 2-3 leaf stage of development (approximately 10 days after sowing), cut 2-3 inches of three leaves per sample and place in a deep 96-well plate. OR collect approximately 75mg (3-5in leaf) of fresh tissue into 1.1ml 96 well plates, folding to about ½-3/4in in size.

Either sample one plant per sample, or 3-4 plants per sample, depending on the generation and experimental design of the project.

Step 3: Seal the 96-well plate with either 8-well stripe tops (for long term storage) or a securely taped kim wipe (for quick freeze followed by lyophilization).
Step 4: Freeze at -80 °C overnight at minimum. OR freeze in liquid nitrogen and freeze dry for 24-48 hours. Alternatively, tissue can be harvested and frozen in liquid nitrogen and stored at -80 C until grinding.

Grinding DNA
Step 0: Reserve time on the Geno Grinder by communicating with James Tanaka about project.
Step 1: Turn on the Geno/Grinder 2010 (switch is back left corner). Allow Geno/Grinder to warm-up for 1min before starting.
Step 2: Adjust the time and rpm settings appropriate for your samples by pushing and turning the knobs.

Tissue Type	Prep Type	RPM	Time
Leaf	lyoph.	1400	1:00
Leaf	frozen	1500	:45
Root	frozen	1350	:45
Crown	frozen	1350	:45
Stem	frozen	1350	:45

Going over the recommended RPMs can result in breaking your block, if this happens clean up your mess! These settings were evaluated for VWR 96-deep well blocks.

Step 3: Unless you are using the freezer blocks, place the orange tray labeled “bottom” flat side down (located ¬¬¬¬drawer below grinder) in the holding tray.
Step 4: Place blocks in orange tray (you MUST have an even number of blocks to even out the machine while running, if you need a height BALANCE, there are some in Lab 411).
Step 5: If you are running more than two blocks the Geno/Grinder can accommodate 4 96-deep well blocks. Place an orange nesting tray labeled “nesting” on top of the two bottom blocks then place the next two blocks in the nesting tray.
Step 6: Squeeze the button of the twist top lid to bring it down to rest on top of the two blocks. Finish twisting the knob to tighten lid all the way (finger tight only).
Step 7: Press the green button
Step 8: Wait for the Geno/Grinder to come to a complete stop and the light in green button to become solid again (if you do not wait it can cause an error on the next run).
Step 9: When you are finished turn the Geno/Grinder off.

DNA Extraction
Step 0: Grind to powder on Homogenizer (Genogrinder).
Step 1: Add 610ul extraction buffer preheated to 65C, mix well and incubate at 65C for 30 min. Mix samples every 10 mins.

Mix well: add a pcr mat on the tubes, mix by inversion + brief centrifuge until dissolved (no compact tissue). If there is compact tissue at the bottom of the tube, it will be okay, you just wont get as much DNA.

Step 2: Remove from waterbath, wipe off excess water and allow samples to cool to room temperature for about 10 mins, remove steel grinding balls and add 250-300ul 24:1 Chloroform/Isoamyl alcohol

Removing balls: pull one strip at a time, being away of broken strips. Hold strip carefully and use a wide magnet (on the side of the hood) to loosen (wiggly) the balls at the bottom of the tubes, and shimmy them up the tube. Usually you can get 6/8. Then go back for other 2.

Step 3: Shake vigorously to form an emulsion
Step 4: Centrifuge at top speed for 15 mins (15 min @ 4000rpm or 5 min @ 13000rpm)
Step 5: If RNA free DNA is desired, add 6ul 5mg/ml RNAse to new tube or plate and proceed, otherwise skip to step 6
Step 6: Carefully transfer the upper phase to a new tube (approx 500ul; 2 sets of 225; Use 8 channel)
Step 7: For RNAse treated samples, give a short centrifuge spin (up to 1000rpm) and allow incubation at 37C for 5 minutes, otherwise proceed to step 8
Step 8: Add 500ul isopropanol and (gently) mix well by inverting 15-25 times (can see DNA form)
Step 9: For RNAse treated samples, proceed directly to step 10. Otherwise, optionally let samples sit in -20C for 5-60 mins
Step 10: Centrifuge at top speed for 30 minutes (30 min @ 4000rpm or 15 min @ 13000rpm)
Step 11: Carefully discard the liquid, making sure pellet does not dislodge
Step 12: Add 500ul of 70% Ethanol
Step 13: Centrifuge at top speed for 15-30 min (15-30 min @ 4000rpm or 5-15 min @ 13000rpm)
Step 14: Carefully discard liquid, again making sure pellet does not dislodge. Allow samples to air dry so that no ethanol remains. This can be done for 20-60 mins at RT, or 15-30 min at 37C.
Step 15: Resuspend DNA in 50-100ul ddH20 or diluted TE (preferred) and mix on low vortex. Samples can be refrigerated immediately or allowed to dissolve @ 37C for about 30 mins.

Extraction Buffer (100ml):

Final Concentration:	Location	Amount/Stock Concentration	1 plate 100mL
ddH2O	by sink	--	52.8 mL
2% CTAB	dry shelf	2g/CTAB	2 g
1.4M NaCl	Shelf premade	28.5ml/5M	28.5 mL
20mM EDTA	Shelf premade	8ml/0.25M	8 mL
100mM Tris-HCl, pH8	Shelf premade	10ml/1M	10 mL
0.2% 2-mercaptoethanol	-20C Rm 416	200ul/2-mercaptoethanol	200 uL
0.2mg/ml Pronase E or Proteinase K	-20C Rm 416	1ml @ 20mg/ml	1 mL

TOC

RNA extraction ¶

SAFETY PROTOCOL:
Hazards:
       Phenol: Toxic if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage Suspected of causing genetic defects. May cause damage to organs through prolonged or repeated exposure. Toxic to aquatic life. Do not breathe dust/fume/gas/mist/vapors/spray. Wash thoroughly after handling. Do not eat, drink or smoke when using this product. Use only in a well-ventilated area. Avoid release to the environment. If exposed or concerned: Get medical advice/attention. You must sign this training sheet stating you understand the hazards involving the use of phenol.
       Chloroform: Hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in case of skin contact (permeator). Has CARCINOGENIC and MUTAGENIC effects. Do not ingest. Do not breathe gas/fumes/ vapor/spray. Wear suitable protective clothing. In case of insufficient ventilation, wear suitable respiratory equipment. If ingested, seek medical advice immediately and show the container or the label. Avoid contact with skin and eyes. Keep away from incompatibles such as metals, alkalis. You must sign this training sheet stating you understand the hazards involving the use of chloroform.
       Isoamyl Alcohol: Hazardous in case of skin contact (irritant, permeator), of eye contact (irritant), of ingestion, of inhalation. Keep away from heat and any sources of ignition. You must sign this training sheet stating you understand the hazards involving the use of isoamyl alcohol.
       Ethanol: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly hazardous in case of skin contact (permeator), of ingestion. Flammable, keep away from heat and any sources of ignition. You must sign this training sheet stating you understand the hazards involving the use of ethanol.
      Centrifuge: Before using a centrifuge, read the centrifuge safety brochure and sign that you have read it.

Protection:

Close-toed shoes are always required when working in the laboratory.
Lab coat and RNase free gloves are required at all times during the phenol-chloroform RNA extraction procedure.
Wear protective gloves/protective clothing/eye protection/face protection when handling Phenol.
When using Isoamyl alcohol & Chloroform: Splash goggles. Lab coat. Vapor respirator. Be sure to use an approved/certified respirator or equivalent. Gloves.
When handling liquid nitrogen, always wear protective eye-wear and protective gloves.

Waste:
Organic waste (all the waste from steps 1- 10) needs to be dumped in a designated organics waste brown glass bottle and disposed of by the official WSU hazardous waste department.
Phenol and Chloroform waste must be disposed of in accordance with federal, state and local environmental control regulations.

Spill Clean-Up:
       Phenol: Exposure to the spilled material may be severely irritating or toxic. Follow personal protective equipment recommendations found in the MSDS. Personal protective equipment needs must be evaluated based on information provided on this sheet and the special circumstances created by the spill including; the material spilled, the quantity of the spill, the area in which the spill occurred, and the expertise of employees in the area responding to the spill. Never exceed any occupational exposure limits. Prevent the spread of any spill to minimize harm to human health and the environment if safe to do so. Wear complete and proper personal protective equipment at a minimum. Dike with suitable absorbent material like granulated clay. Gather and store in a sealed container pending a waste disposal evaluation. Block any potential routes to water systems.
       Chloroform: Small Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Large Spill: Absorb with an inert material and put the spilled material in an appropriate waste disposal. Be careful that the product is not present at a concentration level above TLV. Check TLV on the MSDS and with local authorities.
       IsoAmyl Alcohol: Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an appropriate waste disposal container. Large Spill: Flammable liquid. Keep away from heat. Keep away from sources of ignition. Stop leak if without risk. Absorb with DRY earth, sand or other non-combustible material. Do not touch spilled material. Prevent entry into sewers, basements or confined areas; dike if needed. Eliminate all ignition sources. Be careful that the product is not present at a concentration level above TLV. Check TLV on the MSDS and with local authorities.
       Ethanol: Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an appropriate waste disposal container. Large Spill: Flammable liquid. Keep away from heat. Keep away from sources of ignition. Stop leak if without risk. Absorb with DRY earth, sand or other non-combustible material. Do not touch spilled material. Prevent entry into sewers, basements or confined areas; dike if needed. Be careful that the product is not present at a concentration level above TLV. Check TLV on the MSDS and with local authorities.

PROCEDURE:
DNA-free RNA Isolation For Wheat Seeds (adapted)
Isolation of Total RNA optimized for wheat seeds in small microfuge tubes. Protocol is based an Arabidopsis adaption heavily modified to optimize from Sven Nelson based off of a paper published by Oñate-Sánchez and Vincent-Carbajosa in 2008. For protocol ease, the procedure used for wheat has been left in, and the original protocol with the Arabidopsis notes and more thorough details are found here.

Follow the blue text comments to help you prep ahead of time and reduce total extraction time.

As with any RNA extraction, use extreme caution of RNase contamination. DEPC-treat any chemicals that you prepare. For chemicals like Tris that you cannot DEPC-treat, use DEPC’d water to prepare it. Prepare an RNase-Free zone on your bench where no one will enter and you will never touch with bare hands (do ONCE day before; No need to repeat unless the area has been obviously contaminated or a long time has passed). You can buy fancy wipes or solutions to wipe this area down, but if you don’t have these, a low percentage bleach solution will work to remove RNases. Some sources suggest using SDS in water will denature RNases, but this is not recommended. SDS will partially denature the RNases and then wiping it will spread these still-partially-active RNases around your bench causing more harm than good. Always wear gloves from a fresh box and use other precautions, but remember also that some of this is overkill and we do it to be extra-safe. If you happen to touch a tube with your bare hand do not assume that this means that the experiment has failed and toss the tube. It is likely that one touch will not destroy your sample. Also, there will be instances where you will have no choice but to use a less-than-RNase-Free zone, such as when using the hood or when storing things in a -20ºC communal freezer.

----Day 1----
Sample preparation (grinding tissue):
Grinding the tissue is the most important step of the RNA extraction. You need to get your tissue completely ground without allowing it to defrost. You do not want to see any tissue start to get soggy. Keep applying liquid nitrogen as you grind to keep tissue frozen, this requires some care as the liquid nitrogen can cause the sample to splatter up out of the tube or mortar. Avoid this.

Step 1: Grind 20mg dry seed in liquid nitrogen and get sample into a 1.5mL microfuge tube (label tubes morning of). For wheat seeds, use both method a. or method b. Method A will get the samples/tissue into a fine powder, which are placed into a cooled microfuge tubes. Then method b will grind the samples more thoroughly with the dremel.

a. Use a pre-cooled mortar and pestle (prep day before). Scrape the ground tissue out and place in an eppendorf tube without allowing it to defrost. As best as possible, reduce the amount material lost in transfer from mortar and pestle to tube.

b. Then grind the seed in the microfuge tube that has been pre-cooled in liquid nitrogen (backflip easy open tubes work well, but don’t seal as tightly). To pre-cool: Used a metal block with holes for tubes (from a broken tube incubator). Place this in a styrofoam box and surround with ice (mainly for stability), then flood with liquid nitrogen to cool the block. Place the open tubes in the block and fill with liquid nitrogen (liqN2) until the bubbling ceases. This indicates that they have cooled sufficiently (cool right before grinding). While some liquid nitrogen remains in the tube, carefully add your sample to the tube. You may wish to cover the top with a metal spatula to prevent the splattering liqN2 from ejecting your samples. Allow liqN2 to stop bubbling in the tube and when most of the liqN2 has evaporated, grind with a plastic conical tipped head coupled to a handheld dremel (drill-like tool; charge day before ). Wiping the head with 70% EtOH between samples is sufficient to prevent cross-contamination and RNase-degradation.

There are commercial products available for this, but a household Dremel will work just as well and cost much less. When buying a household dremel, a battery powered model is preferred as the cord can get in the way and contaminate your RNase-free area. In addition to this, I believe the battery powered models tend to be less powerful, the corded models may burn through your tubes. If you have the option of one with a high-low speed switch this will allow you to optimize the speed if you are worried about burning through your tubes. Grinding is pretty much instant, no need to hold the grinding head at the bottom for more than a few seconds. No time for the sample to defrost.
To get optimal yield: (for Arabidopsis seeds) grind a little longer until you see the sample coat the wall of the tube and it appears that it is “wet.” Ensure that the tube is still cool, however, you don’t want to heat up the sample sue to friction. The wetness is most likely the oils from the seed being released. When you grind this way you will need to scrape some seem material off of the grinding tip after grinding, but it will more than double your yield (600ng/µl → 1200ng/µl).

Step 2: Immediately add 550µl Extraction Buffer (prep all buffers and aliquot liquids day before ) and move to the fume hood to add 550µl chloroform. Vortex 10 seconds. (Do not allow sample to defrost before adding the buffer.) You may need to stick a pipet tip into the tube to scrape some material off of the edge if you used the optimized grinding method.

i. For all vortex steps I have the vortex set at the line between 5 and 6.
ii. *Extraction buffer: 0.4M LiCl, 0.2M Tris pH8, 25mM EDTA, 1%SDS iii. If you have more than 4 samples for this extraction, then keep the cooled metal block cold.

Step 3: Move vortexed tube (looks like milkshake) to ice if you will have 4 samples or less to extract; if you will have more than 4 samples for this extraction, then place the first tube in the liqN2 cooled metal block that you used for grinding. Then repeat steps 1 and 2 again with the remaining samples. Place sample #2 and higher in the liqN2 cooled metal block. The samples in the liqN2 cooled metal block will freeze instantly.

Once all samples are ground and in Extraction Buffer and chloroform, proceed to step 4. Step 4 through 10 must be done 1 tube at a time. You will need to make balances for centrifugation, but if you do even 4 tubes in parallel you are likely to see a decrease in yield from the 1st to the 4th tube. Leave the remaining tubes in the metal block (add liqN2 periodically to keep cool).

Take Lunch if 4+ samples are being extracted.

Step 4: Remove the sample that is on ice and vortex briefly to ensure there are no ice chunks present. Move the next sample in line from the liqN2 metal block to the ice box to defrost (should return to a liquid milkshake by the time you need to use it) and spin the sample 3 min at top speed in a tabletop microcentrifuge (≈13,000 rpm) at RT.

Once skilled and familiar with the protocol, you can do 2 samples at a time. So samples #1 and #2 were thawed at the same time, vortexed, and centrifuged together. While sample #3 and #4 are placed on ice to thaw. This is the MAX overlap you should do. Any more may result in error due to time it takes to continue the protocol.

Step 5: Transfer aqueous (top) layer to a fresh tube. Because you will do two subsequent organic:aqueous separations, you can carefully suck the aqueous layer all the way down to the interphase division formed by the cell debris.
Step 6: (in hood) Add 500µl of water-saturated acidic phenol (it is important not to use pH 6.6 buffered phenol for this, instead use pH 4.5). Vortex thoroughly after adding phenol. Add 200µl of chloroform and 8µl of iso-amyl alcohol. Vortex briefly, but thoroughly. (This step is important: if you do not mix thoroughly you will get 3 layers after spinning, if you do you will get 2 layer with a very clear interphase.) Spin 3 min top speed at RT.
Step 7: VERY CAREFULLY, remove the aqueous (top) layer to a new tube. Do this right away, do not let the tube sit. Do not suck all the way down to the interphase. (I usually get about 500µl) You want to avoid getting any phenol carry over at this step. But, phenol is very slightly soluble in water, so a small portion of phenol will have gone into the aqueous phase. In order to remove this we need to do one final chloroform extraction.
Step 8: Add equal volume (for me, 500µl) of chloroform to the tube and vortex thoroughly. (Again, it is important to vortex thoroughly if you want this step to have any effect. We want to pull the last little bit of phenol out of the aqueous layer). Then… either a. or b. below.

a. For best results, use Phase Lock Gel (PLG) tubes (from 5Prime). After vortexing, transfer sample with chloroform to a pre-spun PLG tube (spin morning off). Spin down for 3 min top speed at RT.
b. You can still get great results if you simply spin down the tube at this point (3 min top speed at RT.) (just be more careful in the next step as noted.)

Step 9: Transfer supernatant to a fresh tube. If you have used a PLG tube then you can suck all of the aqueous layer out safely with no worry for organic contamination. If you did not then just be careful when sucking the top layer off, don’t suck down too close to the interphase and don’t let the tube sit before transferring the aqueous layer as phenol can come out into the top layer if it sits too long (although unlikely at this point).
Step 10: Now comes the moment of truth: Add ⅓ volume (for me this was ≈167µl) of 8M LiCl to the tube and invert to mix. If you get a white precipitant immediately then you have phenol contamination. This means you didn’t vortex thoroughly as instructed. Don’t panic, you can still save your sample, but you will get lower yield. But if you do not see an obvious white precipitant (the white will be obvious, very faint cloudiness or schlieren lines are ok) then you will have phenol-contamination free extractions. Precipitate at -20ºC for 2 hours minimum. You will get best yield by precipitating overnight. At this point go back to step 4 and repeat steps 4 through 10 until all samples are at -20ºC. Store for a minimum of 2 hour from last sample.

----Day 2----
Step 11: Spin 30 minutes at 4ºC at 13,000 rpm to recover pellet (cool centri. morning of). A visible brown pellet is a good sign.

a. At the same time, take 10x DNase buffer and from the -20ºC and place it on ice. The buffer will be frozen and needs to thaw before use. Place RNase-free DNase I in a stay-cold container (like Stratagene’s StrataCooler Benchtop Coolers) in the freezer ready to take out when you want to use it.

Step 12: Discard the supernatant and dissolve the pellet in 26µl DEPC-treated water. If pellet is hard to dissolve by flicking the tube (no vortex) then add a 10 minute step at 37ºC to allow it to dissolve more before adding DNase.

If you had a white precipitant in step 11: After the spin you will see the white precipitant at the bottom with the pellet. When removing supernatant suck in some liquid and gently “blow” this at the white precipitant. The phenol will come off in organic bubble droplets fairly easily. Make sure to remove all of these droplets. Unfortunately, you will lose some RNA in this step if you do this, but even if you feel like you have harshly blown everything away and your tube looks like it has no pellet, you will still probably get a fair amount of RNA, maybe in the 40-60ng/µl range. If you are careful, you can still get around 150ng/µl yields consistently this way. After resuspending pellet look for any (very small) organic droplets floating around. If they exist, remove them with a 2µl pippetman. If you are careful, you will still get good quality uncontaminated RNA)

Step 13: For Zymo Research Cat# E1007: Add 6.5µl 10x DNase buffer (thaw on ice morning of) and 3.25µl RNase-free DNase I to each sample (no thaw needed, can use straight from the -20C). Incubate at 37ºC for 30 minutes.

Important: Adjust quantities of Buffer and DNase to the recommendation for the brand of your DNase based on a 65µl reaction.
This step removes any remaining genomic DNA. With only 1µl of DNase I, I still saw some genomic DNA contamination on occasion.

Step 14: Add 470µl DEPC-treated water + 7µl 3M NaAc pH5.2 + 250µl 100% EtOH (fresh bottle, not 95%). Mix well by inverting. (NO VORTEX) Spin 10 min at 4ºC to precipitate carbohydrates.
Step 15: Transfer supernatant to new and final tube. Add 43µl 3M NaAc pH5.2 + 750µl 100% EtOH. Mix well by inverting. Leave at -20ºC for at least 1 hour.

Lunch Break!!!

Step 16: Spin down 20 min at 4ºC (13,000rpm). Save pellet. This is your RNA. If you can see a pellet this is a good sign. If not, it does not mean that you have no RNA.
Step 17: Wash with 70% EtOH: Add ≈100µl of 70% EtOH and let it wash over the pellet, then suck it up right away and discard. Do not try to dissolve the pellet in the 70% EtOH.
Step 18: Air-dry open tubes on bench. (For about 10 minutes, until fairly dry, RNA pellet will not turn white or otherwise change.)
Step 19: Resuspend in 20µl of RNase/DNase-free water (ex: Zymo Cat# W1001-10). Nanodrop and enjoy! (It is NOT recommended to use DEPC-treated water at this step.)

Final notes:

Usual yields are > 1000ng/µl from 20mg of dry seed, ≈1500-2000ng/µl if imbibed.
To check quality → run semi-denaturing gel electrophoresis of ≈400ng of sample.
Even better: Run samples on the bioanalyzer and you can get a RIN score.

Original Protocol from:
Oñate-Sánchez, L., and Vicente-Carbajosa, J. (2008). DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Res Notes 1, 93.

Materials needed:	Keep at:
Extraction buffer: 0.4M LiCl, 0.2M Tris pH8, 25mM EDTA, 1%SDS	RT
chloroform (does not need to be treated with DEPC)	4°C
water-saturated acidic phenol (does not need to be treated with DEPC) - pH 4.5	4°C
8M LiCl	4°C
100% iso-amyl alcohol	4°C
10x DNase Buffer	-20°C
RNase-free DNase	-20°C
3M NaAc pH5.5	RT
100% EtOH (fresh bottle, not 95%; keep this RNase-free)	RT
DEPC-treated water	RT
70% EtOH	RT
RNase/DNase-free water - Zymo Research Cat# W1001-10	RT
Liquid Nitrogen	-

optional but recommended Phase Lock Gel tubes (heavy) from 5Prime 1.5mL microcentrifuge tubes
RNase-Free tips (20uL, 200uL, 1000uL)
Ice, metal tube block, Styrofoam box
Centrifuge (one at RT, one at 4°C)
Pipettes (20uL, 200uL, 1000uL)
optional handheld dremmel

TOC