Bio-Rad C/P Lift Membranes - Manual (Page 5)

11. Rinse briefly in 2 x SSC, 0.2% SDS to remove
cellular debris.
12. Air dry 30 minutes, bake 1 hour at 80 °C.
13. Hybridize (see Section 4).
Section 3
Plaque Screening
1. Cool plate containing plaques for 5 minutes.
2. Continue with steps 2 through 9 of Section 2.
Section 4
Hybrization Protocol
4.1 Prehybridization
1. Seal blotted membrane inside a heat sealable plastic
bag.
5
5 Remove the upper sheets of 3MM filter paper and
orient by puncturing both membranes with an 18-G
needle containing black ink. Take care when
removing the needle not to move the membrane.
6. Carefully peel the membranes apart and place the
C/P Lift membrane onto its selective agar plate with
the colony side up. If desired, additional replicas can
be prepared from the C/P Lift membrane master in
the same manner.
7. Return the master filter to its original plate and store
at 4 °C.
8. Incubate the C/P Lift membrane replica until the
colonies are 0.2-0.5 mm diameter, then transfer the
membrane (colony side up) onto a fresh plate
containing chloramphenicol at 200 µ g/ml and
incubate for a further 6-18 h.
9. Place the C/P Lift membrane, colony side up, onto a
pad of 3MM filter paper saturated with 0.5 M NaOH
for 5 min. Blot the membrane disc briefly on dry
3MM filter paper.
10. Repeat step 9.
4
LIT267B 10/28/98 10:12 AM Page 4