cDNA Library Screening

Nitrocellulose Membrane Washing

Denaturation Solution: 2 minutes (First membrane) – 4 min (Second Membrane)

1.5 M NaCl and 0.5 M NaOH denaturation solution

150 mls 5M NaCl 300 mls 5M NaCl

25 mls 10N NaOH 50 mls 10N NaOH

Q.S. to 500 mls w/.H₂0 Q.S. to 1 L w/ H₂0

Neutralization Solution: 5 minutes

1.5 M NaCl and 0.5 M Tris-HCl (pH 7.5-8.0) neutralization solution

150 mls 5M NaCl 300 mls 5M NaCl

250 mls 1M Tris-HCl 500 mls 1M Tris-HCl

Q.S. to 500 mls w/ H₂0 Q.S. to 1 L w/ H₂0

Rinse Solution: 30 seconds

0.2 M Tris-HCl (pH 7.5-8.0) and 2X SSC buffer rinse solution

100 mls 1M Tris-HCl 200 mls 1M Tris-HCl

50 mls 20X SSC 100 mls 20X SSC

Q.S. to 500 mls w/ H₂0 Q.S. to 1 L w/ H₂0

Dry on Whatman 3MM paper.

Crosslink DNA to membranes using Stratalinker UV crosslinker

120,000 micro Joules of UV energy – (Preset –AutoCrosslink-Start)

OR bake at 80° C for 1.5-2 hours

Store agar plates at 4° C.