1)grow 20ml cells O/N37℃ 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℃

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 at37℃ sonicate

again on ice,solution should become viscous.

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

7)spin 7.5k rpm4℃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℃ 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 MgCl₂1M 1

60mM KCl 2M 30

1% Triton stock 10 add before use

0.5mM DTT 1M 0.5 "

0.5mM PMSF 0.5mM 10 "

5μg/ml Leupeptin 5mg/ml dilute before use

5mg/ml antipain " "

check pH!

GUSHISTOCHEMICAL STAINING

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

2)make up required vol of stain:

for 10ml4℃ 5mg X-Gus

50μl 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/ 500μl stain

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

4)inc o/n37℃in humidity chamber

5)asp,inc 10'in FAA:

for 200ml 4℃ 10ml formaldehyde

10ml HAc

75ml EtOH

H₂O > vol

6)inc 2' 50% EtOH

7)inc 2' 100% EtOH

8)inc 1' H₂O