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TAIR: Arabidopsis Protocols

STANDARD PLANT MOLECULAR BIOLOGY PROTOCOLS

Kindly provided by John Mundy, Institute of Molecular Biology, Copenhagen, Denmark.

Agrobacterium competent cells

Agrobacterium electroporation

Agrobacterium miniprep

Agrobacterium miniprep (phenol free)

Arabidopsis transformation (infiltration)

cDNA probes

Colony hybridization

Concanavalin A Blots

DNA PAGE

E coli competent cells (CaCl2)

E coli competent cells (RuCl2)

E coli transformation

End labelled probe preparation

Filter washing

Fluorography of PAG

Fluorometer/Luminometer Wallac Victor II

Footprint with DNAse1

Gelshift

Genomic DNA from cereal leaves

GST fusion protein purification

GUS histochemical assay

GUS & LUC bombardment assay

GUS luminometric assay

HIS-tagged protein purification

Hormone treatments

Hormones & inhibitors

Hybridization selection of cloned DNA

Immunoprecipitation from in vitro translations

Isotachophoresis probe isolation

In vitro transcription

In vitro translation

Isolation of Lambda DNA by plate lysate

Lambda lysate

Leaf PCR

Maxam Gilbert sequencing

Microsomal preps for membrane proteins

Miniprep - alkaline lysis

NEpHGE Non-equilibrium pH gel electrophoresis

Northern blot in formaldehyde gel

Nuclear extract preparation

Nuclei prep

Nuclear prep from protoplasts

Oligo dT cellulose purification of mRNA

Oligo probe hybridization

Oligonucleotide probe preparation

Phage strains

Plasmid prep with CsCl

Primer extension with synthetic oligonucleotide

Progeny analysis of plants

RNA extraction in GT w/ CsCl spin

RNA large scale from starchy tissue

RNA miniprep 1

RNA miniprep 2

RT-PCR

SDS-PAGE for proteins

Seed sterilization for cereals

Silver staining of proteins in PAG

Soluble protein extraction

Southwestern blotting

T4 polymerase fill in rxn

Western blotting

Whole cell extracts


AGROBACTERIUM COMPETENT CELLS

You should double up this protocol - it is almost the same amount of work and you can thus get some 80 tubes.

1. Inoculate colony O/N in 2 ml YEP + antibiotics at 28C shaker. ABI - 50 KAN & 25

Chlor, gv3101 - 25GEN 2. Transfer O/N culture to 200ml YEP in a sterile 500ml flask and shake at 250rpm until the OD is 0.3 (4-5hrs)

3. Spin in sterile 50ml screw cap tubes 4C 5krpm 10‚. Check to make sure cells are pelleted, if not repeat at higher speed.

4. Aspirate supernatant, resuspend pellet in 20ml ice cold 1mM HEPES pH7 (sterile filtered), respin.

5. Repeat 4. two more times!

6. After aspirating, resuspend pellet in 2ml ice cold 10% glycerol (sterile filtered).

7, ASAP dipense in 40ul aliquots in pre-chilled, sterile eppis, freeze in lN2 and store -70C

AGROBACTERIUM ELECTROPORATION

Safety precautions

1. Never change any other settings than stated while unit is charging (this may damage both the electroporator and power supply)

2. Keep unit away from water in a dry area and away from flammable materials.

3. Never short circuit terminals.

4. Whilst delivering pulse, keep hands away from chamber and cuvette. The result may otherwise be shocking.

DNA preparations

DNA for electroporation must be free of salt, RNA or protein. DNA (in TE buffer) should be first treated with RNase, then twice extracted with phenol/chloroform.

This will remove protein and RNA. To remove salt, EtOH precipitate the DNA and wash twice with 70% ethanol. Resuspend the DNA at 0.4 -1 ug/ml.

Preparing the electroporator

There are two types of cuvettes 1 and 2mm. Most Agro protocols use 2mm (Invitrogen #650009 w/blue lids).

1. Make sure power supply is off.

2. With Charge/Pulse switch of the electroporator in the PULSE position, connect the leads from the power supply to the corresponding coloured terminals on the back of the electroporator.

3. Set Arm/Disarm dial to disarmed

4. Set Capacitance selector to 50 mF

5. Set Load resistance to 200W

6. Turn on power supply

7. Set maximum power to 25 W

8. Set current to 25 mA

9. Set voltage to 1800 V

10. Allow power supply to stabilize (still in pulse position)

11. Select the CHARGE position using the Charge/Pulse switch. After 20-30 seconds, the charging light will glow.

12. Check that voltage meter still reads 1800 V

13. Set Arm/Disarm to the ARMED position and the armed light will glow.

14. Switch the Charge/Pulse to the PULSE position. The pulse light will glow briefly and both the charging and armed lights will go out.

15. Set Arm/Disarm to the DISARMED position (the armed light should be off)

Electroporating

Electrocompetent bacterial cells, YEP media and DNA solutions must be kept on ice before mixing. Note that the following steps should be carried out in under 1' and that you should be wearing glasses and gloves

16. mix 1-2ml DNA (600 ng) with 40ml cells

17. Transfer the DNA/cell mixture to a cuvette on ice avoiding air bubbles by gently shaking the cuvette

18. Dry outside of the cuvette with tissue paper and insert the cuvette into the cuvette chamber with notch facing towards you

19. Close cuvette chamber lid

20. Set Arm/Disarm to ARM (arm light comes on)

21. Set Charge/Pulse to pulse and the pulse light will come on briefly

22. When pulse light is off, set Arm/Disarm to DISARM (arm light comes on) and remove cuvette

23. With DNA/agro mix still in cuvette, add 500ml cold YEP (no antibiotics) and mix solution by gently pitppeting up and down

24. Transfer the cells to an eppi and incubate at 28C for 2-4 hour

25. Leave the electroporator with the switch in the PULSE position

26. Plate 200ml on YEP + antibiotics

27. Incubate at 28C and colonies will appear in 2-3 days

Re-using cuvettes

Fill a used cuvette w/ 0.1M H2SO4 and let stand 15'. Rinse 6x w/ dH20, then 2x w/ 96% EtOH. Store them well-covered in 70% EtOH

AGROBACTERIUM MINIPREP

Agros to be used for plant transformation should be checked for the presence of the Ti plasmid as plant transformation and the analysis of transgenic plants is time consuming. The easiest way to do this is to make an agro miniprep and to use PCR to determine that the cells contain your construct. PCR is necessary here because the Ti plasmid is single copy and you can barely see it on agarose gels.

1. Grow cells overnight in 5 ml LB or YEP with antibiotics.

For pMONs in ABI - 50ug/ml KAN, 50ug/ml Spec, 25ug/ml Chlor

For pBI types in gv3101 - 50ug/ml KAN, 25ug/ml GEN

2. Remove 1 ml cells to two microfuge tube

3. Centrifuge 45 sec and remove the supernatant with aspiration

4. Add 1 ml cells more to both tubes and repeat step 3

5. Vortex the pellet, add 100 l MPS1 solution, vortex again and incubate the tubes at room temperature for 5 min

6. Add 20 l of a 20 mg/ml lysozyme solution, vortex-spin 1 sec and incubate 15 min at 37C.

7. Add 200 l MPS2 solution( freshly made), mix gently by turning the rack 3-4 times and incubate 5 min on ice

8. Add150 l MPS3, vortex for at least 10 sec and incubate 5 min on ice

9. Centrifuge for 5 min and remove the supernatant to new tubes

10. Add 400 l phenol/chloroform/isoamyl alcohol (25:24:1), vortex, centrifuge for 5 min and remove the supernatant to new tubes

11. Repeat step 10

12. Repeat this step with chloroform alone

13. Add 300 l isopropanol and incubate on ice for 10 min

14. Centrifuge for 5 min and wash pellet with 70 % EtOH

15. Dry pellet and resuspend the two tubes in a total of 50 l TE-buffer+RNase, use 2ml for a PCR, freeze the rest.

MPS1 for 50 ml Stock

 	50 mM glucose 		1M 2,5 ml  	10 mM EDTA 		0,5mM 1 ml 
25 mM Tris pH=8.0 1M 1,25 ml

MPS2 for 10 ml

 	0,2 N NaOH  	10N  200 ml  	1% SDS 		10%  1 ml  	H2O      	8,8 ml 

MPS3 for 100 ml

 	5 M potassium acetate   60 ml  	glacial acetic acid  	11,5 ml  	H2O     		28,5ml  

AGROBACTERIUM PROTEASE K MINIPREP (PHENOL FREE)

1) Resuspend pellet from 3ml cultures in 200uls STET

2) Add 10ul Lysozyme/RNase solution and incubate for15min at 37 C.

3) Add 1ul 10mg/ml Protease K solution and incubate at 50 C for 15min.

4) Incubate at 94 C 1min then vortex vigourously 30 sec.

5) Remove gooey supernatant to new tube and add 5uls 5% CTAB. Wait 5min then spin 5min.

6) Remove supernatant and rususpend in 300uls 1.25M NaCl by vigourous vortexing. Spin tubes for 1min and transfer supernatant to new tube with 750uls 96% EtOH + 1mM PMSF. 5min RT, 30min spin.

7) Wash pellet in 70% EtOH+ 1mM PMSF, dry and resuspend in 40uls TE 8.0

Can visualize 10uls in a restriction digest.

ARABIDOPSIS TRANSFORMATION BY VACUUM INFILTRATION

This protocol is modified from Bechtold, Ellis and Pelletier (1993). "In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants". [C.R. Acad. Sci. Paris, Life Sciences 316: 1194-1199].

PLANT GROWTH:

1. Take seeds with a brush and place them into 8cm square pots filled with soil. Don't compress the soil too much and water the pots thoroughly with 2-3 pot-vol to remove excess nutrients. Place 12-16 seeds in each pot.

Place the pots in the cold room for two days before transfering them to the growth chamber. Grow the plants for three weeks in short days (10 hr or less) to get large plants and a greater seed yield. Transfer the pots to long days to induce bolting. Grow plants to a stage at which bolts are around 10 cm tall.

2. Clip off emerging bolts close to rosette leaves to encourage growth of multiple secondary bolts. Infiltration will be done 7 to 9 days after clipping (plants will be 10-15 cm high and the biggest of the inflorescens will have made the first tiny silique. Do not water the plants the day before vacuum infiltration.

VACUUM INFILTRATION:

3. Start a 4ml agrobacterium culture (YEP+antibiotics) inoculated from a -800C stock or from a plate. Grow cells O/N to 48h depending on the strain. Add this culture to 250 ml of YEP+antibiotics (A 250ml culture will give enough cells for infiltration of 6 pots). Grow the culture between O/N and 2 days (depending on the strain) to OD600 = 1.2-1.8. The culture will have a mother of pearl appearance (not lumpy or black).

4. Spin down agros at 5000rpm for 10 min in 250ml centrifuge bottles, resuspend in infiltration media to an OD600 = 0.8 in a minimum volume of 300ml.

5. Poor the agro suspension into a beaker of an appropiate size (400ml is ok). Place the beaker into the vacuum jar. Degass the solution by drawing vacuum until bubles form. Place a paper towel under the beaker to avoid that the beaker gets stuck in the bottom of the vacuum jar.

6. Sprinkle the plants with water 5 min prior to infiltration (optional) and then invert plants into the culture solution. Make sure that all the flowers are submerged and leave 2cm between the rosettes leaves and the agro suspension. Don't let the culture contact the rosette or soil as this could kill the plants. Avoid that the solution boils over when you pull the vacuum. Make sure that the soil is only moist, so that the water from the pots does not enter into the culture suspension (therefore we recommend not to water the plants the day before infiltation). Draw vacuum for 15-20 min for WS and 30 min for Col-0 at a pressure close to 0.05 Bar (we are using an oil pump).

7. Before removing the plants from the vacuum jar place a plastic bag over the pot and beaker. Pull out and remove plants from the beaker, lay pots on their side (to avoid that excess infiltration media runs down into the soil). Fold over the top of the plastic bag and staple them twice. The other possibility is to place the pots laying on their side into a tray and cover the whole box with saranwrap. Put them in a growth chamber for one night. Next day move them to the green house. Put the plants in vertical position and open the bags. Next day get rid off the bags. In case you have the plants in trays: put also the plants in vertical position and use sticks and saranwrap to make a kind of a tend around the plants. Next day remove the plastic. In hot summers, we recommend to give plants a shower after we have placed them in vertical position (the purpose of this is to remove the sugars from the infiltration media which decrease fungal infection).

8. Grow plants for approx. four weeks, keeping bolts from each pot together but separated from neighbouring pots

9. When the siliques begin to turn yellow, place the pot on its side with the plants inside a big envelope. Leave them for one week to dry out and cut off the plants. Let the seeds dry in the envelope and clean them 10 days later (keep all the seeds from one pot together). Store the seeds in the cold room for one week before plating them.

KANAMYCIN SELECTION PROTOCOL

1. Sterilisation of seeds:

aliquot seeds in 15ml falcon tubes (approx 700 seeds/tube, you can estimate the ammount of seeds by first drawing a square plate of 9cmx9cm on a paper and spreading the seeds on it). Add 10 ml of hypoclorite solution. Shake tubes for 10 min. Remove the solution and add 10ml of 70% ethanol. Wait 2 minutes. Discard EtOH and wash seeds 2-3 times with 10ml of sterile water.

Resuspend seeds with 8ml 0.7% top agar (no warmer than 55oC ). 2. Spread seeds onto selection plates (MS+Kan). Dry plates in laminar flow hood until the top agar has solidified.

3. Vernalize plates for two nights in the cold room at 4oC. Transfer the plates to the growth chamber (21oC with continous light).

4. After aprox 7 days transformants should be clearly identifiable as dark green plants with healthy green secondary leaves and roots that extend into the selective medium. Root growth is the most clear maker to identify transformants at an early stages.

To make sure that the transformants are positive transfer them to a new MS+Kan plate and leave them there for a few days (if they turn yellow is because they are faulse positives). Transfer the seedlings to soil.

If you have contamination on your plates at this step, transfer the transformants as early as possible to soil.

5. Grow the plants and collect the seeds.

Infiltration Media

1/2 x Murashige&Skoog salts (SIGMA #5524)

1X B5 vitamines (1ml of 1000x stock) (SIGMA; #G-2519) Gamborg's vitamine powder, to prepare the 1000x stock disolve 11.2g in 100ml water.

5% sucrose

adjust to pH 5.7 before autoclaving

after autoclaving add:

- Benzylamino Purine (BAP), 10 µl per liter of a 1 mg/ml stock in DMSO. By adding the hormone just before use, you can keep infiltration media as a stock for at least one week prior to infiltration.

- we recommend to add 0.01% silwet to the infiltration media to increase transformation efficiency especially for Landsberg and colombia ecotypes. (silwet is from LEHLE SEEDS, cat no VIS-01 VAC-IN-STUFF (silwet L-77)

Selection plates:

1x Murashige&Skoog salts

1% sucrose

adjust pH 5.7 with 1M KOH.

0.7% Difco agar.

autoclave, cool, and add:

1x MS vitamines (SIGMA #M-7150). Take 1ml of 1000x stock prepared by disolving 10.3gr in 100ml of water.

antibiotic (kanamycin 50mg/l).

Top agar:

1x Murashige&Skoog salts.

1% sucrose.

adjust pH 5.7 with 1M KOH.

0.7% Difco agar.

autoclave.

before use: boil in the microwave and keep in water bath at 50-550C.

YEP media (liquid):

10 g /l Bacto peptone (Difco)

10 g/l Yeast extract (Difco)

5 g /l NaCl

For YEP plates add 15gr/l Difco bacto agar.

Hypoclorite solution:

for 50 ml:

4ml Na Hypoclorite 15%

255l Tween-20

water to 50ml

cDNA PROBES

mRNA 1-5ug

H2O 50ul

oligoDt (10mg/ml) 2ul

heat 90C 2', ice

5xRT buffer 20

10mM dA,G,T 2 ea.

1mM dC 2

1M DTT 1

dCTP* 10

RT 6

inc 42C, 1h

+

4M NaOH 15

10% SDS 7.5

0.25M EDTA 19

H2O 8.5

inc 37C 1h

+ 3ul Hac

PCHCl3 ext

separate from free nucs over G-50 column

use106cpm/mlhyb

COLONY HYBRIDIZATION

filter prep: S&S BA 85 Nc

1) lay filters on plates to moisten

2) replicaplate colonies to them

3) grow o/n

4) transfer filters to 3mm paper on saran wrap soaked w/ 10% SDS. This will lyze colonies; they subside slightly, inc 5'

5) trans filter to dry 3mm briefly

6) trans 2x to 3mm soaked in 0.5M NaOH to denature DNA, inc 5'

7) trans to dry 3mm briefly

8) trans to 3mm soaked w/ 1M Tris 7.5 to neutralize, inc 5'

9) trans to dry 3mm

10)trans to 3mm w/ o.5M Tris/1.5M NaCl to bind DNA, inc 5'

11) wash in 1xSSC w/ 1ug/ml Proteinase K RT 1h

12) wash 10' in 1xSSC

13) air dry, bake 80C 2h vacuum

solutions:

 PB 				/ 1l  50% formamide  			500ml 5xSSC   			250ml 20xSSC 200ug/ml ssDNA  		20ml 10mg/ml sheared boiled salmon sperm 5xDenhardts   			100ml 50x 10ug/ml polyadenylic acid 	1ml 10mg/ml 0.1% SDS 			100ml 10% H2O 				to 1l 

 HB				 /1l  same as PB but  100ug/ml ssDNA 10% dextran SO4   		100g, dissolve this first o/n H2O  

CONCANAVALIN A BLOTS

1) electroblot

2) block in Con A buffer (CAB) RT 3h, 5©10ml/lane

3) inc w/ HRP©Con A RT 3h (1ul HRP©Con A/lane in CAB, approx1:2000 dilution

4) wash 3 x 200ml, 30' RT w/ CAB

5) wash 3 x 100ml, 2' RT w/ HRP buffer (HRPB)

6) develope in DMB solution (DMBS) 1©10'

7) wash in H2O to stop reaction

solutions:

 CAB   			[stock]  		ml/1l  0.5M NaCl  		3M   			160 50mM Tris 7.4 		1M   			50 2.5mM CaCl2  		1M   			2.5 1mM MgCl2  		1M   			1 0.5% Tween 20    				5  

HRPB: 10mM Tris 7.5

DMBS:

10mM Tris 7.5, 20ml

5mg DMB

3.3ul H2O2, immediately before use HRPCon A: type 4 labelled, Sigma # L4010. Make 1mg/ml stock DMB: 3,3'©dimethoxybenzidine dihydrochloride Kodak #X8748

DNA PAGE

% gel 3.5 5.0 8.0 12.0 20.0
30% acryl stock 11.6 16.6 26.6 40.0 66.6
H20 76.3 71.3 61.3 47.9 21.3
10x TBE 5.0 > > > >
10% APS 0.7 > > > >
TEMED (ul) 30 > > > >

seal gel w/ 2mls mix (-TEMED) + 2ul TEMED

range of separation

% gel bp
3.5 100-1000
5.0 80-500
8.0 60-400
12.0 40-200
20.0 10-100

migration of marker dyes

% gel BPB XC
3.5 100 460
5.0 65 260
8.0 45 160
12.0 20 70
20.0 12

1)Plates: Long plates for sequencing and oligo preparations, medium for S1s and strand separations, short for gel shifts and restriction digests. Clean plates w/ soap & H2O, rinse w/ deion H2O, then EtOH, wipe dry. Siliconize notched plate, wash excess w/ EtOH. Taping is unnecessary, use 3 sided spacers and plug w/ 2ml gel mix + 2ul TEMED in pasteur pipette.

2) Gels: Acryl stocks are filter sterilized. First warm urea in H2O to dissolve.

Cast gels last 5-7d RT, are made 0.5 x TBE. prerun for sequencing, unecessary for prep gels.

 		[gel]    		6%  g urea  	21.4 ml 10x TBE  	2.14 /ml acryl 	(38/2%)6.43 ml H2O  	18.21 ul 10% APS  	178.5 ul TEMED  	42.84 

3) Samples: loading buffer is:

 		200ul formamide 		12ul 10xTBE 		32ul dyes (0.8% BPB & XLC) 

heat to 95C 2', cool on ice, warm to RT prior to loading

4) Running: attach aluminum plate. At 2000v-2500v, short sequence xc 27cm 2hr, medium xc 65cm 4hr, long xc 110cm 7hr or 1200v o/n

5) Fixing: remove notched plate

leave 15' in 20% EtOH bath

run-off dry

transfer to Whatman 3MM paper

dry at 80C under vaccum

expose w/ screen o/n

5% 6% 8% 12% 16%
g urea 30 > > > >
ml 10x TBE 3 > > > >
ml acryl (38/2%) 7.5 9 12 19 25
ml H2O 27.5 25.5 22.5 15.5 9.5
ul 10% APS 250 > > 500 >
ul TEMED 60 > > > >

  

E COLI COMPETENT CELLS (CACL2)

1) Grow 5 ml culture o/n w/ or w/out antibiotics depending uponstrain

2) Inoculate 500ml media, grow to 0.5OD600

3) Spin 10', 3000 rpm

4) Resuspend in 165 ml 0.1M CaCl2

5) Stand 20' on ice

6) Spin 10', 3000rpm

7) Resuspend in 30ml 0.1M CaCl2, 20% glycerol

8) Freeze in 0.2ml aliquots in LN2

E. COLI COMPETENT CELLS (RUCL2)

Double up the following protocol so you get plenty

The eppis used in step 9 should be pre-chilled at -80C

1. Streak bacteria on fresh plate and grow O/N

2. Pick 5-6 fresh colonies and disperse in sterile eppi w/ 1ml SOB

3. Inoculate 100ml SOB in sterile 1l flask. Grow 37C 2-3hr to OD595=0.2 (low density critical)

4. Spin cells in four 50ml sterile screw cap tubes at 2500rpm 15* 4C

5. Pour off sup, invert tubes briefly to remove excess liquid. Resuspend pellets in 8ml RF1/tube (1/3 vol.)

6. Inc on ice 15‚

7. Spin cells 2500rpm 15‚ 4C

8. Repeat 5 and resuspend pellet in 1ml RF2/tube (1/25 vol.) and ice cells 15‚

9. Aliquot 100ul well suspended cells in eppis and freeze immediately in lN2.

Store at -80C

SOB 500ml (use within 2-3 weeks)

 Bactotryptone 		10g Yeast extract 		2.5g NaCl   			292mg Kcl   			0.9g H2O   			to 500ml 

RF1 100ml

 RbCl   			1.2g MnCl4H2O  		0.99g KOAc   			3ml of 1M pH7.5 (adjusted w/ KOH) CaCl2 2H2O  		15g  Adjust to pH5.8 w/ 0.2M Hac. Filter through 0.22um. Store RT 

RF2 50ml

 RbCl   			60mg MOPS   			1ml 0.5M pH6.8 (adjust w/ NaOH) CaCl2 2H2O  		0.55g Glycerol  		7.5g  Adjust to pH 6.8 w/ NaOH. Filter through 0.22um. Store RT 

E COLI TRANSFORMATION

1) thaw competent cells (-70 C stored) on ice, invert to mix

2) add 150ul cells to DNA samples in 13ml tubes on ice

3) inc 25' on ice with occassional mixing

4) heat shock 5', 37 C

5) inc ice 5'

6) add 1ml LB ‰without antibiotics_, shake 1hr 37 C

7) spin 30', asp to 200ul, plate 100ul, store the rest

For blue/white screen, add IPTG and X-Gal to plates before starting transformation

200ul 100mM IPTG (0.2g to 8.3ml H2O, 0.22 filter

62.5ul 4% X-Gal (0.4g to 10ml DMF, 0.22 filter

store both at -20C, best if aliquoted. Do not mix before use.

Positive control uses 10ng supercoiled plasmid

END LABELLED PROBE PREPARATION

1) Digest plasmid w/ 5'overhang enzyme

2) P/CHCl3 ext

3) EtOH ppt

4) Redissolve to 0.5mg/ml in H2O

5) Start label rxn:

3ul digested plasmid (1ug)
7ul H2O
2ul each a©32P dNTPs
2ul 10x medium salt restriction buffer
1 ul klenow
15' RT

6) Add 2ul imM cold dNTPs

7) 15' RT

8) Add 35ul TE

9) P/CHCl3 ext

10) EtOH ppt

11) To isolate probe, digest w/ 2nd enzyme to free probe

12) Separate fragments by 6% non-denaturing PAGE

FILTER WASHING

1) Remove filter from hyb solution directly into LSWB at RT C

wash 15'

2) Discard & rewash w/ 500ml LSWB at RT C

3) Discard & wash 3x w/ LSWB 15' at 60 C (see below)

5) Check filter with counter. If more than 10, proceed to 5), otherwise to 8)

6) Discard and wash 3x w/ HSWB at 60 C

7) Check filter w/ counter. Should be less than 10.

8) Blot dry briefly, pack in clingfilm. Tape pack in cassette w/ End labelled probe preparation_

1)digest plasmid w/ 5'overhang enzyme

2)P/CHCl3 ext

3)EtOH ppt

4)redissolve to 0.5mg/ml in H2O

5) start label rxn:

3ul digested plasmid (1ug)

7ul H2O

2ul each a©32P dNTPs

2ul 10x medium salt restriction buffer

1 ul klenow

15' RT

6)add 2ul imM cold dNTPs

7)15' RT

8)add 35ul TE

9)P/CHCl3 ext

10)EtOH ppt

11)to isolate probe, digest w/ 2nd enzyme to free probe

12)separate fragments by 6% non©denaturing PAGE (see page. ptc)

screens and phosphorink spot tapes to orient.

9) Insert film (AXR5 Kodak) and store at -70 C till development.

LSWB 2X SSC, 0.1% SDS

HSWB 0.2X SSC, 0.1% SDS

The easiest way to heat these solution is to submerge large, double zipper plastic bags in water bath.

The temperature used varies for different probes and samples. Make sure you know what tempt to use. 60 C is standard for homologous DNA/DNA/RNA hybs.

FLUOROGRAPHY OF PAG

1) fix as usual

2) rinse 2-3x1h or overnight in 50% EtOH

3) incubate 1hr in DMSO in fume hood in metal/glass tray. If the gel starts turning white, put in back in 50% EtOH and wash longer. You can keep reusing the

DMSO, but the gel should see some fresh DMSO each time before it goes into the DMSO/PPO.

4) rinse 2 x 1hr in 20% PPO/DMSO (poisonous), then 1hr in 20% PPO/DMSO with 3% glycerol. These solutions can be kept separately and used in the same order several times.

5) soak in H2O w/ washing 10' to ppt PPO in gel. With gloved hand gently wipe gel surface free of excess PPO. Plenty of water and gentle agitation required here.

6) dry gel on cellulose sheets (Biorad) above thick paper 60C, 2-3h w/ good vacuum. Every geldryer/vaccuum system is different. You need a good setup for this. Handle the dried gel carefully and tape it to the cassette screens. The gels curl like crazy when frozen

7) expose at 70C to prevent swelling

FLUOROMETER/LUMINOMETER WALLAC VICTOR II

Samples for both Glucuronidase (MUG) and Luciferase assays should be prepared according to the "GUS & LUC bombardment assay" protocol.

1. Switch the Victor II and the computer on. Windows 95 launches automatically the "Wallac 1420 manager" program.

2. In the "Tools" menu, check that the "User level" is set on "Advanced". Otherwise, the "Routine" menu would disable any system operation which means that you could not change any setting, even in your own protocols.

3. Select a protocol available in the list in the "Instrument control" menu (then go to step 5) or create your own protocol (see step 4).

4. Creation of a new protocol menu

4.1 Click with the left button (LB) on "Explorer" in the "Tools" menu in order to launch the "Wallac 1420 Explorer" program. By clicking with the right button (RB) on the "Users" folder, you will be able to create a new protol and then name it.

4.2 Open your new protocol (double-LB). This operation starts the "Protocol Editor" window which should be left open all the time you will work with the Victor II. This program enables you to define all the operations to perform (see step 4.4), to select which wells will be measured (step 4.3) and other things such as the format of the saved files, etc.

4.3 Select the wells which should be measured in the "Sample" menu (LB), the default setting is 96 wells. NB: we use the standard 96 wells white microtiter plates.

4.4 Define the operations to perform in the "Measurement" menu. Select measurement "by plate". To add an operation, you can either click (LB) on the small icons displayed on the left or click in the white operation window (RB). The following operations can be chosen : dispense (if injectors installed), delay (to wait inbetween the measurement of two wells), shake, or label (to select the kind of detection : fluorometry, luminescence, ...). To perform a MUG assay, select "label" (LB) then, in the "fluorometry" menu, select the icon represented with a locker "Umbelliferone (1.0s)" (LB). You have now the possibility to select this protocol (double-LB) and use the default Umbelliferone measurement or make a "copy" of this protocol with an other name. Important : you can not change the settings of any default protocol represented as an icon with a locker. If you want to change some parameters (such as the counting time), you have to create a new protocol (by copying a default one). The default "Umbelliferone (1.0s)" protocol is well adapted to our MUG assays.

4.5 Save your protocol which should now appear in the listing of the "Wallac 1420 Manager" window in the "Instrument control" menu. Select it (LB)

5. Run your protocol by pressing "Start". You can have a "Live display" of the measurement. I would recommend to try your protocol once with an empty microtiter plate to check if everything works fine (good connexion with the Victor II, no mistake when you selected which wells should be measured,...).

6. How to run a MUG assay : mix both the extract and the substrate (MUG buffer + methanol) according to the related protocol (GUS & LUC bombardment assay) but do not stop the reaction with CaCO3. Indeed, you can measure the activity at various time (t0, t1h, t2h,...) from the same wells. To do so, incubate the microtiter plate at 37C in an incubator (remember to cover the plate to avoid evaporation). It is recommended, especially when the GUS activity of the sample is totaly unknown, to make a series of dilution (dilute the extract in the extraction buffer) to check wether activities will be proportional.

7. The data will be automatically saved in the same folder than your protocol (classified upon the date). You can open them (double-LB) then export them as an Excel file or other formats.

8. Leave both Fluorometer and computer on during the week. Switch them off during the weekend.

FOOTPRINTING WITH DNASE1

1)probe: same as used for gelshift), isolated by isotacelectrophoresis w/out EtOH ppt which can enature dsDNA

2)probe mix/rxn: volumes x # samples

1ul probe (0.1©0.5ng or 10©20kcpm)

0.15ul 20mM EDTA

0.4ul 10ug/ul dIdC or dAdT (from gel shift assay)

0.5ul H2O

3)DNAse mix: made up near end of binding incubations. DNAse l(Worthington DPFF,Cat

#LS0006330, lot #58A047,5mg) is 1mg/ml in150mM NaCl, 50% glycerol, store at ©20C.

Try 3 different [s] ofDNAse mix (A,B,C)

1,2 & 3ul stock DNAse1

2 ul 1M MgCl2

©> 100ul H2O

4)binding rxn: components titrated & optimized by gel shiftassays

2ul probe mix

Xul extract

©>18ul NEB (see nucprp.ptc)

30' RT

5)DNAse rxn: add 2ul DNAse mix to binding rxn

inc 1' RT

stop w/ 100ul DNAse stop mix:

                    	stock/50ml  6M Urea 		18g 0.4M NaCl           	6.6ml 3M 1% SDS            	5ml 10% 20mM EDTA           	4ml 250mM 10mM Tris 8         	0.5ml 1M 0.8M NH4OAc         	5ml 8M 10ug/ml glycogen       	50ul 10mg/ml 

5)P/CHCl3 ext

6)EtOH ppt

7)PAGE: Resuspend carefully in 8ul sequencing sample buffer (5'vortex, 5' 60C, 1'

vortex, 2' 90C, spin, transfer to new tube,count cpm). Load equal counts on 6% or gradient sequencing gel.

Notes: If extract inhibits DNAse, add 0.1©0.3ul extra DNAse mixto binding rxns.

DNAse requires Mg, some factors are inactivatedby it! Remember ug/KB x 0.66 = picomole thus 1ng of 300bp probe =2 femptomole.

GELSHIFT

1)Nuclear extract: see nucprep.ptc & nucext.ptc extracts should be at least 3ug/ul

Probe preparation: see endlabl.ptc & isotach.ptc. probe shouldbe 10©20k cpm/ul.

Fragments larger than 400bp should not be used. Make A stock (25ug/50ul) of probe plasmid digested at one end w/ 5' overhang.

3)Binding rxn:

1-2ul probe (0.5ng or 20k cpm in isotach 40mM Tris 7.5 buffer)

1-2ul poly dIdC or dAdT (3ug/ul in NEB, sonicated to 300©500bp)

1-6ul extract (5©10ug/ul in NEB)

>10ul NEB (see nucext.ptc)

incubate 30' RT

1ul sequencing dyes immediately prior to loading under tension

4)Titrations: Start w/ extract titration at 3ug/rxn poly dIdC.

At extract [] w/ max binding, do dIdC titration.

work for complete probe binding, none free. Try Mg salts later.

5)Competitions: Fragments should be isolated by PAGE/isotach. 10-100ng DNA/rxn is usually necessary.

6)gels:

Acryl

48.5ml
10ml 30/0.8% acryl stock
1.5ml 10x TBE
50ul TEMED
400 ul 10% APS
run 100©200v w/ circulation

Acryl/agarose gel

H2O80ml H2O
0.7g agarose
boil to dissolve
10ml 10x buffer 100mM Tris 7.5, 10mM EDTA, 30mM NaOAc
10ml 30/0.8% acryl stock
cool to 60C
60ul TEMED
100ul 10% APS
let set for 2hr, prerun with circulation 30' at 100v and run w/ circulation

7)Gels are dried unfixed on Whatman DE 81 sheets at 80C on dryer.Expose o/n -70C w/screen.

GENOMIC DNA FROM CEREAL LEAVES

1)extract nuclei in nuclear homogenization buffer NHB from 1kg leaves (see nuclei prep steps 1-8)

2)resuspend in 35ml TE

3)add 35ml 2x nuclear lysis buffer NLB (see nucext.rcp)

4)add proteainase K to 0.2mg/ml

5)incubate 37C 1hr

6)spin 3k rpm 5' RT

7)add 1g/ml ground CsCl to supernatant

8)spin o/n vti50 45k rpm

9)puncture w/ 21 gauge needle 1cm from bottom, collect & pool viscous fractions

10)dyalize o/n vs. 2l TE in cold room

GST FUSION PROTEIN PURIFICATION

1) grow 20ml cells O/N 37 C, dilute 50X into prewarmed LB, grow to 0.6 OD or about

1hr. Induce w/ 2mM IPTG (238mg/0.5l), grow 3hr, spin 6k GS3 10', freeze at -70 C.

2) extract cells (from 500ml) in 25 mls. Heintz Buffer plus triton (HBT) by gentle pipette resuspending on ice circa 10'after thawing.

3) transfer to 50ml conical ss34 flip top tubes.

4) add 10mg lysozyme powder to the 50ml (cells from 1 liter now in 1 50ml tube), digest 15' on ice.

5) sonicate with large probe 1' 80% power, freeze in lN, thaw at 37 C, sonicate

again on ice, solution should become viscous.

6) add 1mg DNAse and RNAse, incubate on ice 15'.

7) spin 7.5k rpm 4 C ss34 10'.

8) transfer supernatants to conical screw caps, freeze in lN2, may store.

9) Batch adsorb w/ 4ml 50% slurry GT-Sepharose (PL 17-0756-01), 1hr, 4 C, spin 2', 4k on bench.

10) aspirate, resuspend in 25ml HBT, spin, repeat.

11) pour slurry into column (Econo 1.7x20), elute to top, then with 20 column vols. of HB-T.

12) elute protein in minimal volume (5-10ml) HB+5mM GT (Sigma G4251, 1.5 mg/ml).

13) lN2 freeze as 100ml aliquots for GS.

HB 1 liter

[Final] Stock ml/l
25mM HEPES, pH7.9 1M 50
1mM EDTA, pH 8.0 0.5M 2
20% glycerol stock 200
1mM MgCl 1M 1
60mM KCl 2M 30
1% Triton stock 10 add before use
0.5mM DTT 1M 0.5 "
0.5mM PMSF 0.5mM 10 "
5ug/ml Leupeptin 5mg/ml dilute before use
5mg/ml antipain " "

check pH!

GUS HISTOCHEMICAL STAINING

1) determine number of slides needed, multiply by 0.75ml

2) make up required vol of stain:

for 10ml(4C) 5mg X-Gus

50ul nn dimethyl formamide, dissolve

+ 10ml 50mM NaPO4 pH 7

3) sections best cut with vibrating knife

for sections w/ chlorophyll, put in cell-wells w/ 500ul stain

for sections w/out chlorophyll, put directly on slides w/ stain

4) inc o/n 37C in humidity chamber

5) asp, inc 10'in FAA:

for 200ml (4C) 10ml formaldehyde

10ml HAc

75ml EtOH

H2O > vol

6) inc 2' 50% EtOH

7) inc 2' 100% EtOH

8) inc 1' H2O

GUS & LUC BOMBARDMENT ASSAY

1. Bombarded tissue is incubated according to the experiment

2. grind to a fine powdcr with a mortar and pestle in lN2

3. add 500u1 Extract Buffer l00nmM K2HPO4/kH2P04, pH 7.8 1mMDTT. Keep on ice until spin 10' 4C in microfuge. Aliquot about 450ul to a fresh tube. Freeze in lN2 and store -80C.

Fluorometric GUS

Mix pr assay 125u1 2 x MUG amd 50u1 100% Methanol

prewarm at 37C and at t0 add 75ul extract, mix.

When all have been done (up to 50) take out 50u1 aliquots to 250u1 0.3M Na2CO3 prealiquoted into fluoroscan microtiter plates (Microstrips black Ps, cat no. 9502177, Lab systems). These are measured in scanner (Flurorscan II, Labsystems, Finland) and the numbers are t0 values. Remember to include proper - control without bombardment for background (Tback0). Depending upon the levels of activity, generally incubate the rest for 24hr although you can see a slight color change in 2-4 hr if levels are high. Calculate t24-T0 minus Tback24-Tback0. If the values are above 5000, make dilutions as measurements are not linear at this level.

Extract buffer: good for both GUS and LUC measurements

275ml O.2M K2HP04 plus ca. 20ml 0.2M KH2PO4, adjust to pH 7.8, then add 1 vol H20.

Autoclave. Just befrore use add DTT to lmM and leupeptin to 20ug/ml.

2 x MUG: 2mM MUG in 5OmM Na3PO4/Na2HPO4 pH 7, 10mM EDTA, 0.1% Triton X-100, 0.1% Sodium Lauryl Sarkosyl, lOmM DTT. Store in aliquots at -2OC.

MUG:4-methyl-umbelliferyl B-D-g1ucuronide.

Luminometric LUC assay:

1. Mix

20ul lO x (12.5x) LUC buffer

10ul 100mM ATP

1ul 100mg/ml BSA

169ul H20

total is 200ul/special tube for luminometer

2. add 50ul extract, mix

3. Inject 100ul diluted luciferin solution and take measurement after 5-15". This time interval must be constant for all samples!

10 x LUC buffer

250mM Tricine, pH 7.8

150mM MgC12.

lOOmM ATP

in H20 adjust to pH 7.0, sterile filter, store aliquots at -20C

lOmM Luciferin stock:

D(-) Luciferin lOmg (cat 411400 (Boehringer). For 10mM stock, weigh out 1mg (light sensitive!), add 27ul DMSO to solubilize, then add 12ul 3M NaOAc, pH5, mix, then 275ul H2O, Don't make up large amountrs as this is not very stable, even at -80C.

Diluted luciferin solution: 0.5mM in H2O

GUS LUMINOMETRIC ASSAY

GUS-Light is a chemiluminescent reporter gene assay system designed for rapid, sensitive, and non-isotopic detectjon of B-glucuronidase in cell extracts. The GUS-Light reporter gene assay incorporates GlucuronTM chemiluminescent substrate and a proprietary Light Emission Accelerator. The chemiluminescent assay has a wide dynamic range, enabling detecfion of 0.6pg to 2 ng of B-glucuronidase.

The B-glucuronidase detection assay is simple and fast. Cell lysate or purified glucuronidase is first incubated with Reaction Buffer for 1 hour. Glucuron chemiluminescent substrate which is present in Reaction Buffer is catalytically decomposed by the enzyme. The sample is then placed in a luminometer chamber and GUS Accelerator is added which terminates the B-glucuronidase activity and accelerates the emission of light. Chemiluminescence signal intensity is measured as a 5 second integral. The amount of cell extract used in the assay should be adjusted to keep the assay within the linear range. High intensity signals may saturate the detector of a luminometer resulting in artificially low values.

The GUS-Light system has been formulated for luminometers equipped with automatic injectors. Manual injections may be performed, however signal intensities should be measured within approximately the same interval following the addition of GU5 Accelerator to each sample. Reaction components should be scaled down if a luminometer with a smaller volume injector is used, however sensitivity may be affected slightly. Alternate lysis buffGrs mav be used, however we recommend that their performance is compared with the CUS Lysis Buffer to ensure optimum results of the assay. A Lysis buffer compatible with the luciferase assay containing 0.1M potassium phosphate, 1mM DTT, and 1mg/ ml BSA has been tested with equivalent performance to the GUS Lysis Buffer.

Bacterial contamination of plant material will cause high background. Best results

will be obtained with sterile preparations. Chlorophyll in concentrated samples may interfere with the chemiluminescent signal intensity. Therefore, if high levels of chlorophyll are present, several dilutions of extract should be assayed.

II. SYSTEM COMPONENTS Each GUS-light (Cat. No. Bg100) contains rtagents sufficient for 200 tests. GUS- Light (Cat. No. BG30O) contains reagents sufficient for 600 tests. Glucuron Chemiluminescent Substrate A l00X concentrate is diluted in GUS Reaction Buffer Diluent prior to use.

GUS Lysis Solution contains 50 mM sodium phosphate pH 7, 10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100 (Store at 4'C). Fresh B-mercaptoethanol should be added prior to use to a final concentration of 10mM (i).

GUS Reaction Buffer Diluent contains 0.1MNaPO4 pH7.0, 10 mM EDTA (store at 4 C) GUS Accelerator contains a ready to use luminescent accelerator (store at 4C)

III.PROCEDURE FOR B-GLUCURONIDASE DETECTION

Positive Control: Add 1ul B-glucuronidase (20 pg of B-glucuronidase, Sigma G-7896) to mock extract equivalent to the volume of experimental extract used.

Negative Control: Assay a volume of mock extract equivalent to the volume of experimental extract used.

It is recommended that all assays are performed in triplicate (step 6).

1. Grind tissue to a powder in lN2. These can be stored at -80C.

2 Aliquot the required amount of GUS Lysis Buffer. Add fresh B-mercaptoethanol to a final concentration of 10mM. Note that the lysis buffer for GUS/LUC can also be used.

3 Add sufficient volume of GUS Lysis Solution to cover the tissue (250 uL of Lysis Buffer per 25 mg of plant material).

4. centrifuge sample in a microfuge for 2 minutes to pellet debris.

5. Transfer supernatant to a fresh microfuge tube.

6. Dilute Glucuron substrate l00 fold with GUS Reaction Buffer Diluent to make GUS Reaction Buffer. This mixture will remain stable for several weeks if stored uncontaminated at 4'C. It is recommended to only dilute the amount of Glucuron substrate that will be used within a one month period.

7. Warm the volume of GUS Reaction Buffer required for the entire experiment to room temperature.

8. Aliquot 2 to 20uL of individual cell extracts into luminometer sample tubes. If less than 20 uL of sample is used, Lysis Solution should be added to 20 uL final volume. Note: The amount of extract required may vary depending upon the degree of expression and the specific luminometer utilized. Use 5ul of extract for positive controls and 10 to 20ul of extract for experiments with potentially low levels of enzyme. It is important to vary the concentrations of extract in order to record the signal within the linear range of the assay.

9. Add 180 ul of GUS Reaction Buffer to a luminometer tube and mix gently.

Incubate for 60 minutes at room temperature. Incubations may be as short as 15 minutes (especially if high levels of expression are expected), but the dynamic range of the assay may decrease. Note: Light intensities are time dependent.

Reaction Buffer should be added to sample extracts within the same time frame as they are measured in the luminometer. For example, if it takes 10 seconds to complete a measurement. then Reachon Buffer should be added to tubes every 10 seconds.

9. Place tube in a luminometer. Inject 300ul of GUS Accelerator. After a 2 to 5 second delay following injection, count the sample for 5 seconds. If manual injection is used, then the Accelerator should be added in the same consistent time frame as the addition of Reaction Buffer.

REFERENCES

1.Callagher, S.R. ifl "GUS Protocols: Using the GUS Gene as a Reporter of Gene Expression", Ed. S.R. Callagher, 1992, Academic Press, 47-59.

Fulton, R., and B. Van Ness. Luminescent reporter Gene Assavs for Luciferase and B-galactosidase Using a Liquid Scintiliation Counter. BioTechniques 14(5): 762-763 (1993).

3. Nguyen, V.T., M. Morange, and OBensaude. Firefly Luciferase Luminescence Assays Using Scintillation Counters for Quantitation in Transfected Mammalian Cells. Anal. Biochem. 171, 404-8 (1988).

Manufacturer: TRØPIX, Inc., 47 Wiggins Avenue, Bedford, Massachusetts 01730 (617) 271-0045 or (800) 542-2369 FAX (617) 275-8581. Local AH diagnostics 86 101055

HIS-TAGGED PROTEIN PURIFICATION

1 liter cell prep

1) grow 20ml cells O/N 37 C, dilute 50X into prewarmed LB, grow to 0.6 OD or about 1hr. Induce w/ 2mM IPTG (238mg/0.5l), grow 3hr, spin 6k GS3 10', freeze at -70 C.

2) resuspend cells (from 500ml) in fliptop in 25 mls. UPB8.0, freeze lN2.

3) thaw at 37 C, sonicate 30" high, freeze lN2, thaw.

4) spin RT 10' 15k ss34, respin in fresh tube 10' 15k ss34.

5) decant again to new tube, add 5ml 50% Ni-NTA resin, mix gently RT 30'.

6) spin, decant and wash resin 3x in 50ml UPB6.3 w/5' gentle shaking each time.

7) resuspend in 25ml UPB6.3, pour into column and elute to top with another 25ml.s

8) elute bound proteins 20ml w/ UPB6.3+250mM imidazole.

9)lN2 freeze and store at -70 C.

UPB 1 liter

 [final]    		stock   	ml/l  8M UREA    		solid   	480g 10mM TRISHCl 8.0 or 6.3 1M   		10 0.1M Na-PO4		0.2M pHed   	500 1mM bme    		14.27   	70ml 250mM imidazol          solid           0.34g/20ml. 

HORMONE TREATMENTS

For aleurones, place layers from 100 seeds in 9cm petri w/ 20ml

aleurone solution:

10mM CaCl2 (1.47g/l)
1X antibiotic solution

100X antibiotic stock:

5mg/ml ampicillin
10mg/ml neomycin
1mg/ml nystatin

GA 7mg/1ml EtOH, warm at 37C to dissolve, use 20ul/20ml aleurone solution

ABA 5mg/1ml EtOH, ditto

cycloheximide, make 5mg/ml stock in EtOH, use 40ul/20ml aleurone solution

chloramphenicol, make 50mg/ml stock in EtOH, use 40ul/20ml aleurone solution

HORMONES & INHIBITORS

For protoplasts, dilute stock first 100X in media, then 10x to protoplasts in media.

Phytohormones

ABA: [active] 50mM, stock 50mM, MW 264, stock = Sigma A1012 13.2mg/ml EtOH, dilute 1000x

GA: [active] 10mM, stock 10mM, MW 346, stock = Sigma G7645 34.6mg/ml EtOH, dilute 1000x

Me-JA 98% solution, 5ul/1ml EtOH, 100ul/9.9ml Inc. Firmenich Geneva buffer/media, dile 10 or 100x.

Protein synthesis inhibitors

cycloheximide: [active] 20mM, stock 10mM, MW 281, stock = Boe 103675 2.8mg/ml EtOH, dilute 1000x

chloramphenicol: [active] 100mM, stock 100mM, MW 323, stock Sigma C0378 = 32.3mg/ml 50% EtOH, dilute 1000x

anisomycin: [active] 60mM, stock 60mM, MW 265, stock = Sigma A9789 15.9mg/ml 50% EtOH, dilute 1000x

2nd messengers/inhibitors

dibut cA/GMP: [active] 1mM, stock 100mM, MW 469, stock = Sigma B1381 47mg/ml H2O, dilute 100x

Ca ionophore: [active] 5mM, stock 5mM, MW 524, stock = Sigma 7272 2.6mg/ml DMSO, dilute 1000x

PMA: [active] 10mM, stock 10mM, MW 616, stock = 6.1mg/ml DMSO, dilute 1000x

Froskolin: [active] 10mM, stock 10mM, MW 410, stock = 4.1mg/ml DMSO, dilute 1000x

HYBRIDIZATION SELECTION OF CLONED DNA

1a) dsDNA, 50up plasmid DNA denatured and randomlycleaved