Dominik Haudenschild August 11, 1993 at CBRC

1) Electroporate cells and grow overnight on LB-antibiotic plates

2) Make LB-antibiotic plates containing:

50µl 0.1M IPTG

100µl 2% (in DMF) X-gal

Allow to dry in TC hood 30'

3) Take Nitrocellulose colony lift from original plate and place

colony side up onto IPTG/X-gal plate

4) Grow Induced cells on Nitrocellulose 3 hours 37°C

5) Put plate into 4°C overnight to allow Blue color to develop

6) Put filters onto 2ml Lysis Buffer colony side up for 10 minutes

7) Rinse filters by floating on 500ml PBS colony side up for 10 min.

8) Submerge filters in PBS-Tween and scrub lightly with Q-Tip

9) Process filters like western Blot:

10) Pick Antibody positive colonies that were not blue

11) Do 2ml overnight culture and further screen by doing both

westerns and DNA restriction enzyme gels to verify:

a) Translation of protein that matches predicted size

b) Reactivity of antibody with correct sized band

c) Presence of only 1 insert in plasmid

d) Presence of only 1 plasmid

4M Guanidine = 19.1g

5M Urea = 15.0g

pH should be about 7.0 Don't adjust