Protocol - Colony lift for SH3 family

COLONY LIFT FOR SH3 FAMILY

1. For each plate to be lifted, make one induction plate. Trace amts of IPTG are OK for induction, so add 1痞 IPTG stock to 100痞 H2O, spread on plate with appropriate antibiotics.

2. Lay (using gloves) 0.05痠 pore nitrocellulose filter over colonies and push out (w/ forceps) any bubbles. Lift filter (w/ forceps) and transfer to induction plate, colony-side up. Incubate 2 hrs. @ 37 C.

3. During this incubation period, mix 20痞 biotinylated PPII cell lysate with 1痞 SA-AP (in a 1.5 ml eppendorf tube) for each filter to be developed. Add T-TBS to a volume which exceeds 300痞, and is a multiple of the number of filters to be developed. For example, if developing 7 filters, mix 140痞 cell lysate with 7痞 SA-AP, then add 203痞 T-TBS. This gives a total volume of 350痞, 50痞 of which will go to each filter. Mixture should be rocked @ RT ~2 hrs.

4. Wash colonies completely from filter under running dH2O. The H2O does a fairly good job of softening the colonies, but some rubbing with gloved hand may be necessary to remove all colony traces.

5. Block filters in 5 mls T-TBS 10 min, RT/gentle shaking.

6. Add 2痞 streptavidin to each filter, 10 min, RT/gentle shaking.

7. Add appropriate amount of AP-PPII, 2 hrs, RT/gentle shaking.

8. Wash filter 3x w/ T-TBS.

9. Equilibrate filter (~1 min) in 5 ml high pH buffer.

10. Develop filter in same buffer by adding 100痞 BCIP + 100痞 NBT, shaking occasionally. Color will develop anywhere from 1 to 30 min. If filter begins to turn blue-gray, go to next step.

11. Wash filter with dH2O, lay on a paper towel to dry.

Buffer/solution recipes:

T-TBS
0.5% Tween in 1x TBS

High pH buffer
- 100 mM Na2CO3
- 100 mM NaHCO3
- 1 mM MgCl2

or
- 100 mM Tris
- 100 mM NaCl
- 5 mM MgCl2

BCIP is 15 mg/ml in DMF

NBT is 30 mg/ml in 75% DMF

made Feb. 22, 1999; Baker Laboratory

http://depts.washington.edu/bakerpg