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 by boiling

in 0.33 N NaOH 5', cool on ice,
bringto 2M NH4OAc

1b) ssDNA, 250ug ssDNA is boil 5' and diluted to 2MNH4OAc

2) filter onto wetted Nc disks using cutaway filterholder

3) wash w/ 10 vol 1M NH4OAc

4) bake 80C 2h vacuum

5) cut filter into 6©8 pieces w/ sharp razor on parafiˆlm,

careful for flying pieces!

6) inc in 1ml prehyb buffer PB in round bottom 1ml nunctubes o/n

42C

7) aspirate, add in 100ul hyb buffer HB

8) warm to 65C, cool to 42C and add 1ul RNAsin

9) inc 3-6h 42C

10)low stringency wash

2 x w/ 1x SSC, 0.5% SDS, RT, 10'

3 x w/ 0.1x SSC, 0.1% SDS, RT 10'

1 x w/ 0.1x SSC, 0.1% SDS, 50C 10'

high stringency wash if needed

1 x w/ 1x TE, 60C 10'

11) remove filters quickly to eppis, add 300ul H20, boil

12) remove to new eppi on ice, add 50ug tRNA carrier

13) EtOH ppt

14) resuspend in 4ul H20, translate 2ul, load half of this

Filters:S7S BA 85 13mm, 0.45um, Cat # 4020260

Cutaway holder: Millipore filter holder Cat # xx3001200.This unit

has has had one end cut away making it a
funnel.

Solutions: DEPC treated H20, autoclave

PB
stock /2.5ml

50% formamide 1.25ml

10mM Pipes 6.5 200mM 125ul

0.4M NaCl 5M 200ul

4mM EDTA 0.25M 40ul

500ug/ml wheat germ tRNA*

20ug/ml polyadenylic acid 10mg/ml 5ul

0.2% SDS 10% 50ul

*exhaustively PCHCl3 exted

HB
same as PB but

100ug/ml tRNA

Polyadenylic acid

10-20 ul Poly A+ mRNA/filter

IMMUNOPRECIPITATION FROM IN VITRO TRANSLATION

1) count net inc (TCA filters)

2) total product lanes require 100kcpm for 2-3 day exposure. Total
product aliquots can be cleared of charged
globin as follows:

make up to 10 or 20ul w/ TE

add 2 vols sated AmSO4

mix, stand on ice 30'

spin 5' 4C, aspirate sup

resuspend pellet in SDS-sample buffer

3) aliquot 200k-5x 106 cpm for imm.ppt. depending upon antigen.

500kcpm good for starters. Dilute to
600ul w/ Tris-triton buffer (TTB). Other buffers
better for membrane proteins (see Anderson & Blobel,
Methods in Enzymol.
vol 93, 111).

4) + 10-15ul first antibody, usually pre-immune or normal sera

(Dako #902-3) to bind non-specific proteins.

5) inc w/ slow mixing 1h RT

6) + 50ul 1:1 Protein A Sepharose (PAS) in TTB. resuspend before

pitteting with cut-off p200

7) inc 30' RT

8) spin 1' RT

9) transfer sup to new tube, + 15ul 2nd antibody (specific for

antigen), inc 1hr RT.

10) pellet (proteins bound non-specifically to pre-immune serum

and Sepharose) is washed 3 x w/ TTB and 3
x w/ tris buffer (TB). aspirte final pellet
to dryness w/ hamilton, resuspend in 45ul SDS-sample buffer

11) specific antigen in supernatant from step 9 is adsorbed after

1h w/ PAS for 30' as before, then washed and
prepared for SDS-PAGE as before
(steps 7-10)

Note: depending upon antigen, it is possible to continue specific

immunoprecipitations w/ a third (second
specific) antibody. In
this case, the supernatant obtained in step 11 is adsorbed again w/ PAS before
immunoprecipitating out the second antigen

Solutions:

PAS swell 1h in TTB before use

TTB /l

50mM Tris 7.5 50ml 1M

150mM NaCl 50ml 3M

10mM Methinonie 1.5g

2% Triton 20ml

TB

10mM Tris 7.5 10ml

SDS-sample buffer (see SDS-PAGE)

ISOTACHOPHORESIS PROBE ISOLATION

1)assemble plastic column pieces

2)pack with Sephadex G50 fine running buffer

3)equilibrate with 40mM Tris pH 7.5 buffer

4)secure dialysis tubing with a luer ring or slice of tubing

5)assure there are no trapped bubbles!

6)remove top buffer to bed top

7)load gel slices on top

8)add 5ul sequencing dyes to slices

9)overlay w/ 100mM 6©aminocaproic acid (Sigma A2504)

10)stick column bottom into lower Tris buffer reservoir

11)connect up to power as shown

12)run at 3mA (100-300v)

13)current may vary, adjust voltage occassionally

14)knock bubbles from upper(©) electrode occassionally

15)stop current when dye is 1.5cm from bottom,

16)replace caproic acid w/ Tris buffer

17)remove dialysis tubing and elute DNA, 2 drops/eppi

DNA elutes before dye so careful!

columns

Kontes #220160©0000 col. comp. poly 200mm pkg/100

Kontes #420162©0000 poly bed support filter pkg/100

dialysis tubing

Spectropore cutoff 3500 in TE, no azide!

IN VITRO TRANSCRIPTION

see Contreras et al. (1982) NAR 10: 6353-63 and Stratagenes Bluescript
(pSK) protocols. Stratagene's in
vitro transcription kit with protocols
works fine. Use CsCl purified plasmids with T7 and/or T3 promoter(s).
ORF should
have a 5' ATG with a good ribosome-binding site (Joshi (1987) NAR 15: 6643-53)
and a 3' stop
codon. If plasmid lacks T3/7 terminator, linearize at 3' end of
ORF with a 5' overhang enzyme 0CHCl3 ext.,
and EtOH ppt.

5 ul 5x TB (250mM Tris 7.9, 30mM MgCl2, 10mM Spermidine, 50mM

NaCl)

X ul water to 24 ml total

1-5 ug digested plasmid DNA

1 ul 10mM rATP

1 ul 10mM rUTP

1 ul 10mM rCTP

1 ul 0.1mM rGTP

3 ul 2mM 7mGpppG (Pharmacia 27 4635 01)

1 ul 0.75M DTT

1 ul RNAsin (40U/ml)

1.5 ul T3 or T7 Polymerase

inc 37 C 5', then add

1 ul 10mM rGTP

inc 37 C 20'. This mix can be frozen away for extended periods
and aliquots used to program in vitro
translation. For RNA probes, DNAse the plasmid,
0CHCl3 and EtOH ppt.

IN VITRO TRANSLATION

Use Promega's nuclease-treated -Met (L4210) or -Cys (L4240). The

lysate is the same, just the amino acid
mixes differ. The Promega protocols
work fine as follows, although it is often best to scale them down.

35 ml lysate

7 ml H2O (50 ml final depending on mRNA)

1 ml RNAsin

1 ml 1mM amino acid mixture

2 ml mRNA (1 mg or less)

4 ml Met or Cys (1200Ci/mMol, 10mCi/ml)

inc 37 C 60', place on ice

spot 1 ml on Whatman 1MM, air dry on tin foil

boil in 10% TCA 10', sink filters with ice, rinse 2 x quickly
in tap water, once in EtOH and once in acetone,
air dry. Count with 5ml Econofluor.

SDS-PAGE ANALYSIS

Take desired volume of mix and dilute to 20 ml w/ water. Add 40ml

of cold, saturated AmSO4 (66% final),
mix, spin and inc on ice at least
30'. Spin 10' at 4 C, aspirate supernatant completely off and resuspend pellet
in SDS sample buffer. This ppt. reduces background significantly. Follow SDS-PAGE
protocol (see note for
Cys translations).

LAMBDA DNA BY PLATE LYSATE

for 4 large plates, 50-100kpfu/plate:

1) pool 12ml o/n washes

2) spin out cells 10k, 15' RT

3) + to sup: 20ul DNAse 1 (10mg/ml) and 50ul RNAse (10mg/ml)/

10ml sup

4) inc 30', 37C

5) transfer to 150ml corex bottles

6) + 1 vol 2M NaCL/20% PEG

7) inc 30' on ice

8) spin 7k 20' 4C

9) drain & wipe well

10)resuspend in 5ml TE
11)transfer to 15ml corex

12)+ 150ul .25M EDTA, 150ul 10% SDS

13)+ 0.5ml Proteinase K (10mg/ml)

14)inc 65C, 20'

15)P ext

16)PCHCl3 ext

17)CHCl3 ext

18)EtOH ppt -70C

19)resuspend in 4ml TE, add 4g CsCl, 100ul EtBr

20) spin in small quickseal vTi65 55k o/n

Plates are: NZY for 1 liter

5g NaCl

2g MgSO4 7H20

5g yeast extract

10g NZ amino acids (gibco tryptone, casein hydrolysate peptone

140)

15g difco agar

top agarose is for 200ml

1.6g NaCl

1.2g agarose litex

2g tryptone

LAMBDA DNA BY LIQUID LYSATE

1) Add cored single plaques (1 plaque = 107 phage) to 0.5ml fresh

cells (ON culture in LB w/ 10mM
MgSO4 & 0.2% maltose) in 50ml copolymer
tubes. Make also mock - plaque.

2) inc. 15' 37C

3) add 5ml LB + 5mM CaCl2

4) Shake hard 37C 4-5hrs until cleared w/ cell debris -compare

to control

5) add 100ul ChCl3, shake 5' 37C

6) spin 3000rpm table top 10'

7) transfer sup to corex, add 4ml 20% PEG,2M NaCl, invert well,
inc 60' on ice.

8) spin 3000rpm tabletop 20min, drain and wipe tube dry.

9) resuspend pellet in 750ul LB, transfer to eppi, add 750ul LB/DE52,

invert 20-
30x.
10) spin 5', transfer sup, spin again, transfer sup.

11) add 7ml sup 13ul 0.1mg/ml Poteinase K, 32ul 10% SDS, mix inc
RT 5'

12) add 130ul 3M KOAc, inc 88C 20', cool on ice 10', spin 10'
4C

13) transfer 800ul and add 800ul -20C isopropanol, inc -70C 15',
spin 10'

14) decant well and briefly speedvac pellet, resuspend in 20ul

TE.

LB/DE52

Wash (mix/settle/aspirate) 50g Whatman DE52-cellulose in 100ml

blue cap with 0.05

N HCL till pH under 4.5. Add 10n NaOH dropwise w/ stirring till
pH 7. Wash 3x w/ LB. Add LB to 25%
final vol w/ 0.2% NaN3. store 4C

LEAF PCR PROTOCOL

(Klimyuk et al., 1993, TPJ 3: 493-494)

1) Samples are harvested in 1.5 ml tubes and stored on ice.

2) 40ul of 1N NaOH was added and the samples boiled for 30 sec.

3) 40ul of 0.25N HCl then 20ul Tris mix was added and the samples
boiled for another 2 min.

-tissue samples can then be used immediatly or stored at 4 C for

several weeks.

-The amount of tissue used in each PCR reaction should not exceed
2mm2 or the reaction will not work. A
small amount of treated material can
be excised for use in a PCR reaction with a sterile Gilson tip.

PCR reaction conditions are as follows:

total volume= 50ul

for 5.5 reactions

10X buffer 5ul 27.5uls

10mM dNTPs 1.25uls 6.875uls

primer A 2.5uls 13.75uls

primer B 2.5uls 13.75uls

dH2O 38.75uls 213.1uls

taq poly 1.0ul 5.5uls

95 C 10min 1X

95 C 30sec

55 C 30sec

72 C 45sec 30X

72 C 10min 1X
run 15ul on a 2% agarose gel

note: 2.5 times more primer is used and 2 times more taq polymerase
in the leaf PCR protocol. If you could
get by with less, Jonathan Jones would
have done so!

Stocks

0.25N HCl

0.25N NaOH

Tris buffer:

0.5M Tris pH 8.0

0.25% Nonidet P-40

LEAF PCR ON ARABIDOPSIS TISSUE WITHOUT ALKALINE TREATMENT

Preparation of Master mix:

1 x Taq-buffer

1.5 mM MgCl2

200 mM of each dNTP

1 mM each primer

0.5 ml 20 x Taq polymerase

The mix is stored on ice until use.

Preparation of leaf tissue:

Put the leaf in a small Petri-dish. Make a hole in a leaf with

the narrow end of the Pasteur pipette (a forceps
might be helpful) and place
the leaf in a PCR-tube, if necessary by blowing. On ice, add 50 ml the Master
mix.

Running the cycles:

Transfer the tubes directly from ice to the prewarmed 94 C block

on the Robocycler and run the following
cycles:

94 C for 3 min 1 x

94 C for 30 s; Tann.* for 1 min; 72 C for 1 min-1 min 30 s

35 x

72 C for 10 min (optional) 1 x

Appropriate controls:

Positive Negative

For screening transgenics: plasmid ColO

ColO with endogenous primer-set gDNA

gDNA with endogenous primer-set - DNA

*The annealing temperature (Tann.) should be 2-3 C below the

calculated Tann..
MAXAM GILBERT SEQUENCING

NEN kit #NEK010

1)Modification reactions: volumes are in ul, DNAs are 10©20kˆcpm/ul

Do G, then T&C and C while A&G

is incubating

G react A&G react T&C react C react

200 G buf 10 H2O 10 H2O 15 5M NaCl

5 DNA 10 DNA 10 DNA 5 DNA

1 DMS 2Pip For 30 hydrazine 30 hydrazine

2', RT 15', 37C 5', RT 5', RT

50 DMS stop snapfreeze 200 hz stop 200 hz stop

750 EtOH 750 EtOH 750 EtOH

5' spin 5' spin 5' spin

2)Cleavage reaction: speedvac all samples

resuspend in 100ul 1M piperidine (diluted from 10M stock)

inc 30', 90C, tighly sealed

3)Cleanup

+ 1ml butanol, vortex

spin 3', aspirate

+ 150ul 0.1% SDS, vortex

+ 1ml butanol, vortex

spin 3', aspirate

+ 500ul EtOH

spin 3', aspirate

speedvac

4)PAGE, see ureapage.ptc

MICROSOMAL PREP

from Halkier & Møller 1989 Plant Physiol. 90, 1552-9

1) 0.1-1g sample freeze-dried material powdered in motar/pestle

under lN, suspended in 20xv/w MEB
(microsome extract buffer):

250mM sucrose

100mM Tricine (7.9)

50mM NaCl

2mM EDTA
2mM DTT

100mg/20ml PVPP (BDH Polyclar AT or Sigma 6755)

0.8mM PMSF

5ug/ml antipain & leupeptin

2) Spin 10' 10,000g

3) Transfer supernatant w/ pippette to black double capped polycarbonate

tubes

(16x76mm, Cat # 355603). Save pellet.

4) Spin supernatant 60' at 165,000g (That's 70krpm in a 70ti).

5) Pippette off supernatant and save.

6) Resuspend microsomal pellet in 0.1-1ml MEB. Dyalyze against

50mM Tricine (7.9)

2mM DTT

0.8mM PMSF

MINIPREP - ALKALINE LYSIS

1) 1.5ml o/n culture to eppi, spin 1', aspirate sup

2) resuspend by vortex 5' RT in 100ul miniprep solution 1 MPS1

3) + 200ul MPS2, invert tubes rapidly 3x, inc 5' ice

4) + 150ul MPS3, vortex upsidedown 10", inc 5' ice

5) spin 5' RT

6) transfer to eppi - 7a) for sequencing

7) PCHCl3 ext

8) spin 2' RT

9) transfer eppi

10)+ 900ul EtOH

11)inc 2' RT

12)spin 5' RT

13) aspirate

14) 70% EtOH wash & spin

15) aspirate, speedvac

16) resuspend in 50ul TE, use 2 for digests

7a) +900ul EtOH

8a) spin 5' RT

9a) aspirate

10a) + 1mL 70% EtOH, spin

11a) aspirate, resuspend in 200ml TE, 2mg RNAseA, inc 15' 37 C

12a) Phenol/CHCl3 extract, add 20ml 3M NaOAc, EtOH ppt, 70% wash

13a) resuspend in 30ml TE

14a) see sequenase protocol for denaturation

16) resuspend in 50ul TE

solutions:

MPS1, frozen stock /50ml

50mM glucose 2M 1.25ml

10mM EDTA 0.25M 2ml

25mN Tris 8 1M 1.25ml

MPS2, fresh /10ml

0.2N NaOH 10N 200ul

H20 - 8.8ml

1% SDS 10% 1ml

MPS3 /100ml

60mM KOAc 5M 60ml

1.2M HAc [] 11.5ml

H2O - 28.5ml

NEPHGE NONEQUILIBRIUM PH GEL ELECTROPHORESIS

1) Gel tubes are 0.25 x 13.5cm, acid (1/1 H2SO4/HNO3) washed,rinsed,

silated, rinsed EtOH, H2O, baked,
one end plugged w/ 2xparafilm

2) make gels afternoon of day before electrophoresis

3) make up gel solution

4) polymerized w/ 20ul fresh 10% APS, 7ul TEMED

draw into syringe fitted with 23 gauge and PE tubing, add frombottom
to top.

Aspirate solution back down to 1.5cm from top toclear meniscus

5)overlay w/ H2O 1©2h while polymerizing, replace w/ 20ul

samplebuffer o/n, parafilm top
6)secure dialysis tubing soaked in lower buffer to bottom of gelsw/
tubing slice.

Avoid bubbles, best to do this w/ end submergedin lower reservoir

7) aspirate sample buffer from gel top

8) load sample (10ul), cover w/ 10ul overlay buffer, upper bufferto

top

9) connect © electrode to bottom, electrophorese 400v for

2400Vh

10) aspirate upper buffer, then overlay, then sample, rinse topof
gel w/ sample buffer, then fill to top w/
SDS©sample buffer

11) expel gel from tube slowly using tubing fitted syringe intofalcon
tubes w/ 5ml SDS©sample buffer.

12) equilibrate w/ rocking 1.5h

13) Load on 7.5-15% SDS-PAG (notched plates) w/ sample buffercontaining

1% pre©electrophoresed
agarose. MW standard wells arecast on
left side with plastic teeth. Include BPB in some of theagarose.
Electrophorese at 20mA,
16h

Sample prep: salt is not allowed!

lysate & wgerm, 5ul lysate proteins, 20©100kcpm

protein, 50©200ug

solutions:

Gel, fresh

[final] [stock] /10ml

urea 9.2M 5.53g

NP©40 2% 10% 2ml

Acryl/Bis 4% 28.4/1.6% 1.33ml

Amphol 3.5-9.5 1.86% 40% 465ul

Amphol 5.0-7.0 0.14% 40% 36ul

methylamine 10mM 7.8ul

Ampholines are LKB 1818©101 & 1809©121

Sample buffer, frozen 1ml aliquots

urea " " "

NP40 " " "

amphol 3.5©9.5 " " "

amphol 5.0©7.0 " " "

methylamine " " "

DTT 10mM 1M 0.1ml

Overlay buffer, frozen 1ml aliquots

urea 8M 4.81g

amphol 3.5-9.5 0.2% 40% 50ul

methylamine 10mM 7.8ul

SDS©sample buffer

glycerol 10% 10ml

Bme 5% 5ml

SDS 2.3% 10% 46ml

Tris 6.8 2.5M 0.5M 12.5ml

Upper buffer

36.6M, 2ml/l

Lower buffer

83.5mM ethanolamine, 5ml/l

NORTHERN BLOT IN FORMALDEHYDE GEL

gel prep:

1) add 2g agarose to 143ml H20, microwave, put at 60C

2) + 20ml 10x northern running buffer NRB at 60C

3) + 37ml formaldehyde at 60C

4) cast well mixed in fume hood

sample prep:

1) mix RNA + H20 4.5ul

10x NRB 2ul

formaldehyde 3.5ul

formamide 10ul

2) heat 60C 15'

3) quickly + 2ul/northern sample loading buffer NSLB

4) quickly load

5) run 2 h 100-150v in 1x running buffer NRB

6) when run complete, soak gel 1h in 20x SSC

7) transfer to Nc in 10x SSC

8) prehyb 6h, hyb 40h, 42C in PB & HB (see colony hybridization)

9) wash filters 2x 55C, 1xSSC,1% SDS, 20'

Nx 55C, 0.1xSSC, 0.1% SDS, until low background

solutions
NRB, autoclave, turns yellow

0.2M Mops 7

50mM NaOAc

5mM EDTA

NSLB, DEPC H2O, filter sterilize

50% glycerol

1mM EDTA

0.4% BPB

0.4% XC

MW markers:BRL RNA ladder Cat # 5620SA

NUCLEAR EXTRACT PREPARATION

Based on Parker & Topol (1984) Cell 36 357©369, &

Green et al.(1987) EMBO J. 6 2543©2549) using
swelling in low osmoticum andlysis at high
osmoticum. AmSO4 ppt concentrates and separateshistones from
binding proteins. All
steps in cold room on ice.Volumes here for 100mg nuclear protein (see nucprep.ptc).

1) thaw frozen nuclei on ice

2) add 1 vol NWB triton, transfer to 15ml corex

3) spin 5' 3krpm

4) decant supernatant, drain well

5) resuspend in 7ml total of nuclear lysis buffer NLB, transferto

Beckman ti70

polycarbonate tubes (16x76mm, Cat # 355603)

6) add 770ul 4M AmSO4

7) rock 20', should see viscosity increase

8) spin 1h, 35krpm ti70. The pellet is hardish, supernatantviscous

w/ DNA

9) add 0.3g/ml AmSO4 to supernatant w/ stirring in a 25ml beaker.It

takes at least

0.5h to dissolve

10)spin 15', 10krpm in 15ml corex. The supernatant containshistones,

the pellet primarily binding proteins.
Decant thoroughˆly, proteins
in supernatant can be ppted w/ 80% AmSO4

11)add 250ul nuclear extract buffer NEB to pellet,stand on ice5',

gently resuspend and transfer to dialysis
tubing (cut©off14kD, no azide)

12)dialyze 4h x 2 x 2l NEB

13)spin 10', 12krpm, l N2 freeze supernatant and store ©70C

14)yeild for pea is 5-10% of nuclei prep protein, 2-5% for rice
&wheat. Good extracts are 5-10mg/ml

Solutions: autoclaved, precool, add DTT, Bme and Pase inhibitorsprior

to use

4M AmSO4, BRL enzyme grade

NLB Stocks/100ml

110mM KCl 3.7ml 3M

15mM Hepes/KOH 7.6 7.5ml 0.2M

5mM MgCl2 500ul 1M

1mM DTT 100ul 1M

5ug/ml antipain & leupeptin 100ul 5mg/ml

NEB

40mM KCl 1.2ml 3M

25mM Hepes/KOH 7.6 12.5ml 0.2M

0.1mM EDTA 50ul 0.2M

10% glycerol 10ml

1mM DTT 100ul 1M for resuspension

5ug/ml antipain & leupeptin 100ul 5mg/ml for resuspension

5mM Bme 30ul for dialysis

0.8mM PMSF 1ml 80mM for dialysis

NUCLEI PREP

Based on Watson & Thompson (1986) Methods Enzymol. 118 57©75,Green

et al. (1987) EMBO J 6
2543©2549, and Kannangara et al.(1977) Carlsberg
Res. Commun. 42 431©434.

Set up cold room daybefore, all solutions at 4C, all tubes on

ice.

1) 2.5l (15-20 trays) of seed is grown 7 d in the dark (givesless

starch) in vermiculite w/ occassional H2O.
Harvest w/ scisˆsors to yeild
approx. 1kg leaves.

After weighing, leaves arefurther cut to approx. 2 cm length.

2) wash half in 4l H2O

3) rinse w/ 1.5l homogenization buffer HB

4) transfer to large razor blender, add 2l HB

5) smash w/ 3 1" bursts, then 15" low speed

6) filter thru 1000u and 80u nytex screens (see diagram)

7) rinse blender w/ 500ml HB, brush lower screen to free debris

8) spin 15' (time to smash other half of material) at 3k rpm in
8250ml swing©out fuge tubes

9) decant & resuspend gently (only wide©mouth pipettes)

in 5ml/tˆube (80ml total) nuclei wash buffer NWB
+ Triton, transfer to 430ml Corex tube
and wait for next smash

10) spin corex tubes 5', 3k rpm in swing©out

11) resuspend gently with small brush in 10ml NWB Triton/tube(40ml

total)

12) spin 5', 3k rpm

13) for storage, resuspend in 4ml NWB Triton, mix w/ 1.6mlglycerol,freeze

in lN2 and store at -70C

14) take 1 & 5ul for Bradford assay. Yeild for rice approx
80-120mg, twice that for pea

Solutions: autoclaved, precool, Bme and Pase inhibitors addedprior

to use
Homogenization buffer HB
stocks/l

1M hexylene glycol(Aldrich) 127.5ml 7.8M

10mM Pipes/KOH 7 10ml 1M

10mM MgCl2 10ml 1M

0.5% v/v triton X©100 25ml 20%

5mM Bme, 300ul

0.8mM PMSF 10ml 80mM

Nuclei wash buffer NWB

same as HB but

0.5M hexylene glycol

w/ or w/out triton

NUCLEAR PREP FROM PROTOPLASTS

1) 8x106 protos in cellwells, collect and spin in 2x 15ml APM

at 500rpm for 5' in bench fuge to pellet.
decent supernatant. Subsequent steps at
4 C. Examine cells here, and after next step to check lysis.

2) Lyse by resuspending in 50ml ice cold NIB.

3) Filter through a 20m screen, rinse sreen w/ 50ml NIB, pool

in 3x 30ml corex tubes, 1 tube balance.

4) Pellet nuclei in HB4 swingbucket at 2470rpm (1000g), 20',

4 C.

5) Gently resuspend in 40ml NIB, pool in 2x 30ml corex.

6) Spin to pellet starch and debris in HB4 at 110rpm (1g), 3',

4 C.

7) Transfer supernatant containing nuclei to new tubes. Pellet

at 1750rpm (500g)

15'. Decant completely, quickly wipe tube dry.

8) Resuspend nuclei in 5ml HEN-5. Transfer to ultra tube.

9) Add 0.55ml 5M NaCl ([0.5M] final) and 27.5ml 1M spermidine

([5mM] final).
10) Gently agitate at 4 C, 2hr.

11) Spin clear in Ti50 rotor at 37.5k rpm (88kg) for 15', 4 C.

12) Transfer supernatant to fresh ultra tube. Add 0.45g/ml finely

crushed AmSO4, gently agitate at 4 C for
30' after AmSO4 fully dissolved.

13) Pellet in Ti50 at 28k rpm (50kg) for 30', 4 C.

14) Resuspend in minimal volume HEN-20. Try 50ml, followed by

50ml to rinse tube.

15) Dialyze in small tubes against 2 x 100ml HEN-25 for 2x 3hr.

17) lN2 freeze in 20ml fractions, store at -70 C.

NIB stock ml/l

10mM TRIS-HCl 7.5 1M 10

10mM KCl 2M 5

10mM MgCl 1M 10

10mM ßme 14.27M 700ml

20% Glycerol 100% 200

0.02% Triton X-100 100% 200ml

HEN stock ml/l

20mM HEPES-KOH 7.5 0.5M 40

1mM EDTA 250mM 4

50mM NaCl 5M 10

5-20% Glycerol 100% 50-200

1mM DTT 1M 1

OLIGO DT CELLULOSE PURIFICATION OF MRNA

Cat # 20003, oligo(dT)©cellulose type 3, Collaborative research

Inc., 128 Spring

St. Lexington Mass 02173

Column prep: suspend cellulose in elution buffer, wash in disˆposable

spin columns set in falcon tubes w/ 10
volumes binding buffer

Binding buffer: 10mM Tris 7.5

500mM NaCl

1mM EDTA

0.5% SDS

Wash buffer: same SDS

Elution buffer: 10mM Tris 7.5

1mM EDTA

OLIGO PROBE HYBRIDIZATION

Tm = 4/GC + 2/AT or (IN)

Hyb buffer (OHB)

6 x SSC

10 x Denhardt's

50ug/ml ssDNA

0.1% SDS

Hyb at Tm-10C w/ 2 x 106 cpm/ml

wash: RT 20' 6 x SSC x 2

Tm -5 5' 6 x SSC x 1

RT 5' 6 x SSC x 1

OLIGONUCLEOTIDE PROBE PREPARATION

use #b/A260 = ul containing 100pmole to determine []

if probe has not be desalted or gel purified, spin 1-5 OD260 u

thru 3ml G-50 column, washing w/ 0.4ml
aliquots, pool most []ed 3

A)

1) dry down 100pmole in speedvac

2) add 1ul 10x buffer: 700mM Tris 7.6

100mM MgCl2

1mM KCl

50mM DTT

and 1ul 660uM gATP

1ul 10mM spermidine 7

5ul 32-P dATP

1ul T4 kinase

1ul H2O

3) incubate 30', 37C

4) separate DNA from 32-P dATP by 16% urea PAGE (12% if >30b)

5) visualize bands by autoradiography

6) isolate DNA by isotachophoresis (see isotach.ptc) or crush
and soak (see crshsoak.ptc)

b)

1) dry down 150uCi gATP

2) add 1.5ul 10x buffer: 500mM Tris 7.5

100mM MgCl2

1mM Spermidine HCl, pHed

1mM EDTA

50mM DTT

and 12ul H20

1.5ul kinase

3) inc 60', 37C

PHAGE STRAINS

XL1-Blue: grow in LB/12.5mg/ml TET, 0.2% Maltose, 10mM MgSO4

Plate on same media/top agarose

IPTG: 5ml 0.5M stock per ml top agarose

X-Gal: 16.6ml 250mg/ml stock/ ml top agarose

1088-90: grow in LB/100mg/ml AMP, 0.2% Maltose, 10mM MgSO4

Plate on same media/top agarose

IPTG: 10ml 0.1M stock per ml top agarose

X-Gal: 10ml 2% stock per ml top agarose

PLASMID PREP WITH CSCL

1)grow o/n in 500 ml lb in 2l flask w/ good aeration

2)spin cells 5.5k, 10'

3)pour off, towel dry all supernatant

4)resuspend in 6ml cold sucrose buffer SB, no bubbles!

5)transfer to tubes for Beckman ti 70(Nalgene 3118.0028) or fixed

angle low speed screw cap tubes

6)add approx. 15mg lysozyme

7)vortex

8)stand 5' on ice

9)add 7.5ml triton buffer TB

10)stand 5' on ice w/ mixing

11)spin 30k, 30' in ti70 or 20k, 1h in fixed angle low speed

12)decant all supernatant to screw cap 50ml tube

13)measure volume and add 1g/ml ground CsCl

14)fill to 36ml with 1g/ml CsCl in TE

15)add 0.2ml 10mg/ml EtBr

16)add 2ml 20% Lauryl sarcosine

17)transfer w/ 50ml syringe w/ 16 gauge needle to vti50 quickseal

tubes (Beckman # 342414), seal

18)spin in vti50 49k rpm o/n

19)harvest lower band to 30ml corex

20)extract w/ = vol CsCl/TE sat'ed isopropanol

21)spin 3k, 5', discard top, repeat until clear

22)add 1 vol H2O, divide into two tubes, and add 2 vol EtOH each

23)-20C ppt. 3hrs

24)spin 7.5k, 15', 0C

25)70% EtOH wash & spin

26)resuspend in 5 ml TE

27)EtOH ppt. again

28)resuspend in 0.3ml TE

a good yeild is 1mg plasmid DNA

solutions:autoclave but for isopropanol, precool SB & TB

Sucrose buffer SB stock/100ml for 12 preps

50mM Tris 8 5ml 1M

50mM EDTA 8 20ml 0.25M

15% sucrose 15g

Triton buffer TB

50mM Tris 8 5ml 1M

50mM EDTA 8 20ml 0.25M

0.4% Triton 0.4mltriton

1g CsCl2/ml TE, 400mls

isopropanol sated w/ CsCl/TE, 400mls sated w/ 100ml 0.5g CsCl/ml

TE
PRIMER EXTENSION WITH OLIGONUCLEOTIDE

all solution 0.1% DEPC treated & autoclaved

1)purify and kinase 200ng oligo (see oligoprp.ptc and oligokin.pˆtc)

10ul oligo

10ul 32PgATP

3.2ul 10 x kinase buffer

2ul T4 kinase (2u)

7ul H2O

inc 30', 37C

2)test incorporation by DE81 or TLC (see inctest.ptc)

3)PCHCl3 ext

4) coEtOH ppt w/ mRNA and carrier tRNA (PCHCl3 pure), as follows:

tube # 1 2 3 4

oligo 20ng 7.5ng 1ng 1ng

mRNA 1ug > > 0

tRNA 10ug > > >

5)resuspend in 25ul primer annealing buffer PAB, transfer toscrew

cap eppi and parafilm

6)place in boiling H2O bath 2'

7)transfer quickly to 90C H2O bath, reset to Tm-15C (previouslydetermined

for bath). Tm defined:

Tm = 16.6log[Na] + 0.X(% G+C) + 81.5 © 500/#b of oligo

at 0.4M NaCl, 1% formamide decreases Tm 0.65C, and 1% mismatchdecreases

Tm 1.5C

8)leave to anneal at Tm several hr

9) EtOH ppt w/ 0.2 vol NaOAc

10) resuspend in:

2ul reverse transcriptase buffer RTB

2ul 10mM dNTPs

0.5ul 14.5 ul H2O

1ul BSA

14.5ul H2O

1ul MMV RT (BRL) or AMV

11)inc 1h, 37C

12)1ul 200mM EDTA

13)PCHCL3 ext

14)EtOH

15) run sequencing gel on products depending upon extension length

solutions:

PAB RTB, see BRL catalogue

0.4M NaCl

40mM Pipes 6.9

1mM EDTA

PROGENY ANALYSIS OF PLANTS CARRYING 35S SENSE/ANTISENSE GENES

1. Transform plants by infiltration, grow to seed (F1). Transgenes

should be dominant (expressed
constitutively), therefore phenotypes possible
in these seeds although there are so relatively few
transformants that
they would be virtually impossible to see.

2. Plate F1 seeds on KAN and select for transformants. Examine

these plants carefully during their growth.
Transfer the plants to soil and
grow to F2 seed. The plants are hemizygous (heterozygous for transgene).
Now is the time to take leaf material for Southerns and northerns/RNAse protection.
The former are
performed on DNA digested, for example, with one and
with two polylinker enzymes. Northerns for foreign
genes require only random-primed
DNA probes. To measure sense/antisense transcripts, strand-specific
probes are required. A protocol is described in Curent Protocols (4.7.1)
and good kits are available from in
vitrogen, or Stratagene. Collect F2
seed of all F1 plants. Seed from plants showing good mRNA
accumulation and/or
single copy integration are most interesting. The alleles look like this:
all hemizygous

Rr

R RR Rr

r Rr rr

3) Plate F2 seed on KAN: 25% should be sensitive (rr), 75% resistant

(Rr or RR). Of the 75% resistant, 66%
should be hemizygous, 33% homozygous.
These F2 resistant individuals should be studied for phenotypes,
and various
assays can be performed with them. Transfer to soil and grow these plants
to F3 seed. The alleles
coming out of these plants should look like
this:

66% hemizygous 33% homozygous

RrRR

R RR Rr R RR RR

r Rr rr R RR RR

4) Plate 50 F3 seed from each F2 line on KAN and score sensitive/resistant.

F3 single copy homozygotes should all be resistant. These are

the most important lines for further crosses.
Homozygous, KAN-resistant
plants can be transferred to soil and used for crosses.

RNA EXTRACTION IN GT W/ CSCL SPIN

1)freeze 15©20g material l N2, grind to powder

2)add powder to 50ml flip©top tubes containing 30ml guanidiniumthiocyanate
buffer GTB, shake to assure
that thawing material isunder buffer

3)inc 10', 50C

4)spin 15', 15krpm

5)pipette off supernatant to 50ml screw cap tubes and add 0.15gCsCl/ml.
Do not worry about small amounts
of material floating around

6)layer solution carefully onto 8ml 5.7M CsCL (w/5ul EtBr stock)cushions
in sw28 open tubes. Tubes
should have been inced w/0.1MNaOH/1%SDS for
30', then rinsed thoroughly in H2O pror to use

7)spin sw27 22h, 24krpm 18C

8)aspirate top solution w/ interface junk to cushion, then quickˆly

aspirate cushion around red RNA pellet

9) immediately add 3ml sarcosyl/urea SU, transfer to 15ml corex.vigorous

pipetting required to dissolve
gelatinous flakes

10)add 1.5ml P to fully dissolved RNA, vortex, then 1.5 ml CHCl3,vortex

11)spin 5', 7.5krpm

12)back ext PCHCl3 phase w/ 1.5ml SU, vortex, spin, add SU phaseto
first SU phase

(4.5ml total)

13)EtOH ppt, ©20C

14)resuspend in 100ul DEPC©treated H2O

15)2ul for OD 260/280. ratio should be about 2 for good RNA

GTB, filter sterilized stock/500ml

5M guanidinium thiocyanate 295g

25mM Na citrate 3.67g

0.5% sarcosyl 12.5ml 20%

2mM EDTA 4ml 250mM

5% Bme 25ml

5.7M CsCl/0.1M EDTA 84.185g CsCl/100ml

7M urea/2% sarcosyl 42g urea,10ml 20%sarc/100ml

RNA LARGE SCALE FROM STARCHY TISSUE

1) determine fresh weight of frozen sample

2) grind in lN2-cooled mortar, assure total destruction.

3) mix 1:1 RNA extraction buffer (REB) with phenol, pre-heated

to 50 C.

4) add powder to REB/phenol (1g/2ml) in chloroform resistant centrifuge

tubes. Mix well with polytronuntil
powder dispersed Shake another 5'.
5) Add 1ml chloroform per g sample, shake 25' RT

6) spin 20,000xg, 20', RT

7) transfer top layer to new tube. Back extract interface with

0.5 ml REB/g sample, and repeat 5)-6) on this
and first supernatant.

8) combine supernatants, reextract with phenol/chloroform, spin

10,000, 15' 0 C.

9) For Polyphenol-rich material, add 2ml 50% PolyClar AT in DEPC

H2O/ 100ml extract, then chloroform
extract.

10) measure volume of final supernatant, add 0.1 vol 2.5 M NaOAc
and 2.5 vol EtOH.

mix well, ppts. O/N -20 C

9) spin 10,000, 20', 0 C

10) wash pellet w/ -20C 70 EtOH

11) spin 10,000, 15', 0C

12) decant carefully, dry wipe tube, resuspend pellet in GTB,

pursue rnalrgprp.ptc

13) resuspend RNA in 20 ml. GTB, rinse tubes, transfer to 50ml

screw cap. re-rinse with 8 ml GTB, add
0.15g CsCl/ml. Do not worry about small amounts
of material floating around. Final volume should be 30ml.

6) layer solution carefully onto 8ml 5.7M CsCL (w/5ul EtBr stock)
cushions in sw28 open tubes. Tubes
should have been inced w/ 0.1M NaOH/1%SDS for
30', then rinsed thoroughly in H2O pror to use.

7) spin sw27 22h, 24krpm 18C

8) aspirate top solution w/ interface junk to cushion, then quickly

aspirate cushion around red RNA pellet

9) immediately add 3ml sarcosyl/urea SU, transfer to 15ml corex.

vigorous pipetting required to dissolve
gelatinous flakes

10)add 1.5ml Phenol to fully dissolved RNA, vortex, then 1.5 ml

CHCl3, vortex

11)spin 5', 7.5krpm

12)back ext PCHCl3 phase w/ 1.5ml SU, vortex, spin, add SU phase
to first SU phase (4.5ml total)

13)EtOH ppt, -20C

14)resuspend in 100ul DEPC-treated H2O

15)2ul for OD 260/280. ratio should be about 2 for good RNA

REB, autoclave stock/500ml

100mM Tris, pH 9.0 50ml 1M

100mM NaCl 13ml 3M

10mM EDTA 100ml 0.5M

1% SDS 50ml 10%

5mM DTT 2.5ml 1M

GTB, filter sterilized stock/500ml

5M guanidinium thiocyanate 295g

25mM Na citrate 3.67g

0.5% sarcosyl 12.5ml 20%

2mM EDTA 4ml 250mM

5% Bme 25ml

5.7M CsCl/0.1M EDTA 84.185g CsCl/100ml

7M urea/2% sarcosyl 42g urea,10ml 20%sarc/100ml

RNA MINIPREP 1

1) grind 0.5-2g material in lN2 to fine powder

2) pour 6 large spatulafulls into 30ml corex tubes w/ 10 ml RNAextraction

buffer

REB

3) inc in H2O bath 5©10', 50C w/ occassional shaking

4) add 1.4ml 3M KCl,vortex, inc on ice 10'

5) spin 5', 5krpm fixed angle

6) decant sup thru miracloth to 30ml corex containing 5.6ml 8M

LiCl

7) leave o/n at 4C

8) spin 7.5krpm, 10', 4C

9) dissolve pellet in 5ml H2O

10)PCHCl3 ext

11)EtOH ppt ©20C

12)dissolve pellet in 0.4ml H2O & EtOH ppt ©20C

13)resuspend in 50ul H2O

14)2.5ul for OD260

solutions:

REB stock ml/l

50mM Tris 8 50ml 1M

300mM NaCl 100ml 3M

5mM EDTA 20ml 0.25M

2% SDS_ 200ml 10%

autoclave, then add

1mM aurintricarboxylic acid 10ml 100mM (S#a1895)

14mM Bme 1ml

8M LiCl

RNA MINIPREP 2

0,5-2g plant material

Solutions

REB buffer

50mM tris pH8

300mM NaCl

5mM EDTA

2% SDS

Autoclave then add to aliquots to final concentration:

1mM Aurintricarboxylic acid

14mM Mercaptoethanol

ex. aliquot 50ml gives 500ul aurintricarboxylic acid+50ul mercaptoethanol.

Stocks

100 mM Aurintricarboxylic acid (sigma pH8 w. NaOH)

3 M KCl

8MLiCl

3MNaOc,pH6

Grind tissue (0,5-2g) with pistil/mortar w. liquid nitrogen to

a fine powder.

results are very dependent of this step, so grind well.

1.Add powder to an ELKAY 0002059 tube containing 4,5ml REB buffer

2.vortex

3.+0,7ml KCl

4.Ice 15min

5.spin 10-20min 8k rpm

6.transfer supernatant to a new tube.

7.+2ml 8mLiCl
8.let stand o/n or 5h at 4c

9.spin 8k rpm 20min.

10.Take pellet. resuspend in 2ml water depc. treated

11.vortex.

12.add 2ml phenol/chloroform/isoamylalkhol (25:24:1)

13 vortex

14.spin

15.10min 8k rpm

16.take upperphase to a new tube.

17..repeat 12-15

18.add 200ul 3M NaAc, mix

19.add 5ml 100% EtOH

20.8k rpm 20min

21.wash pellet in 2ml 80% ETOH

22.dry pellet

23.add 100-200ul depc water (2g of tissue gives 200ul water)

24.transfer to a epp. take 4-5ul forOD 260

End -80c

RT-PCR

Total RNAs should have been isolated with a protocole compatible

with the RTase

step (chloroforme extraction following a phenol extraction for

instance).

Contamination of RNAs by genomic DNA should not be too important

(almost invisible on a gel). The
DNase treatment is performed with a RNase-free
DNase in the RTase buffer. Prior to the RTase step, the
Dnase is heat inactivated
and therefore does not require a phenol/chloroforme extraction.

1. DNase treatment of total RNAs

On ice, mix the following :

RNAs 10 ug (not more than 29.5 ul)

10xRTase buffer 5 ul

RQ1 DNnase 2 ul

H20 up to 36.5 ul

Mix well, spin down and incubate at 37 C for 15‚

Inactivate the DNase at 65 C for 10‚ then transfer the tubes
on ice

2. RTase (synthesis of the first strand of cDNAs)

on ice, add the following to the DNase-treated RNAs :

0.1 DTT 5 ul

10 mM dNTPs 5 ul

0.5 ug/ul oligo-dT 2 ul

MMLV-RTase 1 ul

RNase-inhibitor 0.5 ul

Incubate the tubes 1 hour at 37 C then 2‚ at 92 C

For long storage, store the cDNAs at -80 C, otherwise at -20 C.

3. PCR

Perform the PCR by using 1 ul of cDNAs for a 50 ul PCR reaction.

The optimal number of cycles should be
determined in order to stop the reaction
during the exponential part of the amplification, before reaching the
plateau
phase, otherwise the PCR will not be quantitative. Remember to include
appropriate positive and
negative PCR controls.

SDS-PAGE FOR PROTEINS

PA stock made from biorad 2.6% C, add 362 ml. H2O/150g mix. This
gives 30:0.8%.

sterile filter.

gradient gel 7.5% 15% standard 12.5%

30% Acryl stock 11.5ml 22.5ml 37.5ml

resolving buffer 5.6ml 5.6ml 11.2ml

60% sucrose 3.8ml 13ml -

10% SDS 450ul 450ul 900ul

H2O 28ml 4ml 40.0ml

TEMED 15ul 10ul 25ul

10% APS 0.18ml 0.18ml 360ul

5% stacking gel

30% Acryl stock 5.0ml

stacking buffer 7.5

10% SDS 300ul

H20 17.1ml

TEMED 40.0ul
10% APS 0.4ml

8x Resolving buffer, filter, 4 C

363g Tris/l, pH 8.8

4x Stacking buffer, filter, 4 C

60.5g Tris/l pH 6.8

5x Running buffer, 4 C

30.25 g Tris/l

144 g Glycine/l (not HCl salt)

sample buffer, frozen

[final] [stock] /50ml 2x (for dil.)

Tris, 8.8 0.2M 1M 20ml 20

sucrose 0.5M - 18g 36

EDTA 5mM 0.25M 2ml 4.0

methinone 1% - 0.5g 1g

BPB 0.05% - 2mg 3mg

SDS 2.5% 10% 12.5ml 2.5g

DTT 5mM 1M 250ul 500ug

for samples containing cysteine or free 35-S cysteine, add

iodoacetamide 50mM 0.5M 2.5ul/25ul sample

inc 15' 37C, then + 1ul Bme to quench iodoacetamide

2M urea may be used in SDS gels. Allow urea to dissolve fully

before adding SDS.

SEED STERILIZATION FOR CEREALS

1) wash 10' with tap H2O

2) wash w/ 4xV w/ 0.2% AgNO3

3) wash 2x w/ sterile H2O

4) wash 1x w/ 100mM NaCl

5) wash 3x w/ sterile H2O

6) plate w/ 100ug/ml chloramphenicol

100U/ml Nystatin

OR

1) wash 1' 96% EtOH, aspirate

2) wash 10' 30% commercial bleach w/ 1ul/ml 20% TritonX100

3) wash 5x w/ sterile H2O

4) plate as above

SILVER STAINING OF PROTEINS IN PAG

adapted from Boulikas et al. (1981) Anal Biochem 118, 197©203

1) fix gel 30' in fixer, then rinse 2 x 1' in 50% EtOH

2) shake 15' in solution C

3) rinse 5' in H2O w/ rapid shaking and 1 flip of gel. Careful,

gel swells

4) develope in solution D. Careful, stop reaction before gel is

as dark as you want it as development
continues briefly in fixer

5) stop development by washing in fixer

solutions:

A 2g AgNO3 in 10ml H2O

B 54ml 0.1N NaOH + 3.5 ml [NH3]

C add A to B dropwise with stirring. dilute to 250ml w/ H2O. Mustbe

used immediately

D 2.5ml 1% citric acid + 0.25ml 37% formaldehyde. Dilute to 500mlw/

H2O. Must be used immediately

SOLUBLE PROTEIN EXTRACTION

1) grind tissue quickly in chilled mortar in 3ml soluble proteinextract

buffer

(SPEB)/gm tissue

2) transfer to 15ml corex, spin 12k 10' fixed angle

3) trans sup to 15ml corex, add 0.25 vol 50% TCA, chill 4C 30'

4) spin 1k 5', gently pour off sup, do not let pellet dry!

5) + 5ml 80% cold 80% acetone

6) sonicate in cold, spin 1k, 5', aspirate, repeat 3x

7) store protein as acetone suspension ©70C in 1ml 80% acetone

determine [protein]

OD 280: take 10ul to 1ml 0.1N NaOH/1% SDS

Bradford: dry 10ul acetone suspension, dissolve in 10ul 0.1NNaOH,

+

+ 3ul 1M Tris 8, + 10ul 0.1N HCl, dilute to 1ml w/ H2O, use 10-100ul
for Bradford

Solutions:

SPEB [stock] ml/50ml

50mM Hepes 0.5M 5

5% Bme - 2.5

1x BAM/ECAP 100x 0.5

1x PMSF 100x 0.5

H2O - 41.5

100X BAM/ECAP: 100mM benzamidine & 500mM e-amino caproic acid

in H2O

100x PMSF: 80mM/EtOH, ©20

SOUTHWESTERN BLOTTING

Silva et al. (1987) PNAS 84: 1744-48

1) run SDS-PAGE and blot as for westerns

2) place NC in blocking buffer (SWBB), inc RT 1h

3) aspirate and + denaturing buffer (SWDB), inc RT 1h

4) asp and + renaturing buffer (SWRB), inc RT 1h

5) asp, transfer to sealbag, + plenty of SWRB, inc 4C o/n

6) asp and + 10-15 ml binding buffer (SWBiB) + milk

7) asp, + 3 ml (< as possible) SWBiB + milk + DNA probe, inc

RT 1h

8) wash 3 x 5' in SWBiB. expose up to 1 week

Note:

a) milk may inhibit binding. Try 10mg/ml BSA (boiled 10' in 10mM
KHPO4 & 50mM Hepes Ph 7.5) in
SWBB. If milk is thus replaced, omit from SWBiB.

b) [NaCl] in SWRB and SWBiB can be optimized

c) Other variables - Buffer (Tris, Hepes), MgCl2 (Mn, Zn), EDTA,

NP-40

Solutions:

SWBB 5% Non-fat dry milk

30mM Hepes 7.5

SWDB 50mM Tris 8.3

50mM DTT

1mM EDTA

6M Gua-HCl

SWRB 50mM Tris

1mM EDTA
2mM DTT

0.5M NaCl

10% glycerol

0.1% NP-40

SWBiB 30mM Hepes 7.5

50mM NaCl

5mM MgCl

2mM DTT

0.25%Non fat dry milk

T4 POLYMERASE FILL IN RXN

1) resuspend 40ug DNA in 90ul H2O for bal 31 deletions (see baldel.ptc)
Use less

DNA, smaller volumes for normal fillins

2) add 10ul 10 x T4pol buffer

3ul T4 polymerase (BM #1004786100)

3)inc 5' , 37C

4)add 5ul 2mM dNTPs

5)inc 10', 37C

6)add 3ul Klenow enzyme (BM #997463500)

7)inc 5', 37C

8)P/CHCl3 ext.

9)EtOH ppt.

10x T4 pol buffer

330mM Tris-acetate pH 7.9

660mM K-acetate

100mM Mg-acetate

WESTERN BLOTTING

electroblotting: assure that gel is cut to correct size for S&S

BA85 or Millipore GSWP 0.22um membrane
after they have been washed 10' in transfer
buffer. Transfer for 3h at 250mM at RT

immune assay:use 50ml solutions/12x20cm piece

1) wash wet membrane (most antigens withstand membrane drying)

in PBS + 0.5% tween

2 x 30'.
2) aspirate, inc in 1st antibody diluted 500-5000x in 1% BSA-PBS
1-3h

3) asp, wash 3 x 10' in PBS/tween

4) asp, inc in 2nd antibody diluted 1-10k x in PBS/tween 2h

5) asp, repeat 3)

6) asp. for phosphatase wash 5' in 50mM Tris 9.6, 5mM MgCl,asp,
inc in stain A stain solution until products
clearly visible

7b)for peroxidase, inc in stain B

8) asp, rinse 2 x 5' in PBS

solutions:

Transfer buffer, 4C

1 x SDS-PAGE running buffer/20% MeOH

BCIP (5-bromo, 4-chloro indoxyl phosphate), frozen 5mg.ml DMF

NBT (nitro blue tetrazolium), frozen 0.5%/EtOH

PBS/l

20ml M/15 KH2PO4 (9g/l)

80ml M/15 Na2HPO4 (9.5g/l)

30ml 5M NaCl

H2O > 1l

stain A, fresh

50mM Tris 9.6

5mM MgCl2

100ul BCIP

100ul NBT

Stain B, fresh

10mg 3amino-9ethylcarbazole(Sigma A5754)/1.25ml DMF

25ml 50mM NaOAc 5

12.5ul 30% H2O2

WHOLE CELL EXTRACTS

1l rice seed imbibed o/n, grown in dark 6d in 6 trays vermiculite

All steps at 4C on ice

1) harvest w/ scissors, weigh (yeild should be approx 200g), thencut

to 1-2cm length
2) wash in 1l H2O

3) smash in 250ml whole cell extract buffer WCEB, 8 burst 10"

each

4) filter thru 1000u & 80u screens

5) measure volume, add 1/10 volume 5M NaCl, rock 30' 4C

7) spin in quick seal ti70 1h, 40krpm

8) add to supernatant 0.3g/ml ground AmSO4 and 0.1ml 1N NaOH/10gAmSO4

9) stir in small beaker 30'

10)spin 20', 10krpm in swingout 250ml tubes

11)resuspend pellet in 6ml nuclei extract buffer NEB

12)dialyze (cutoff 14kD, no azide) against 2 x 2l NEB

13)spin 15', 12krpm in 15ml corex

14)measure 1ul for Bradford protein. Yeild should be approx 50mg/ml

15)aliquot as 0.5ml, l N2 freeze, store at -70C

solutions: autoclave, precool, add DTT, Bme & Pase inhibitors

priorto use

WCEB stocks/l

40mM Tris 7.5 40ml 1M

5mM MgCl2 5ml 1M

0.5M sucrose 362g

10mM Bme 300ul

0.8mM PMSF 10ml 80mM

NEB

40mM KCl 13ml 3M

25mM Hepes 7.5 125ml 0.2M

0.1mM EDTA 500ul 0.2M

10% glycerol 100ml

1mM DTT 1ml 1M for resuspension

5ug/ml leupeptin & antipain 1ml 5mg/ml for resuspension

5mM Bme 300ul for dialysis

0.8mM PMSF 10ml 80mM for dialysis