Bio-Rad C/P Lift Membranes - Manual (Page 4)

2.2 Replica Lifts (with amplification)
1. Place a C/P Lift membrane disc on the appropriate
selective agar. Plate cells containing recombinant
plasmids onto this membrane, and incubate until
colonies are 1-2 mm diameter.
2. Remove the disc and place it on two clean sheets of
3MM filter paper, colony side up.
3. Place a circle of C/P Lift membrane onto the surface
of a selective agar plate for pre-wetting. Then
transfer it onto the membrane disc with colonies.
Place the surface of the C/P Lift membrane that did
not come in contact with the agar onto the
membrane disc with colonies. Hold the membrane at
opposite edges with forceps, bending it slightly into
a ‘U’-shape, and then lower the membrane until the
fold makes contact with the center of the
nitrocellulose membrane. Lower the edges until the
entire membrane is evenly wetted.
4. Place two clean sheets of 3MM filter paper on top of
both membranes and apply firm, even pressure with
a plate.
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Lower the edges until the entire membrane is evenly
wetted.
3. Orient the membrane and underlying agar by
puncturing both with an 18-G needle containing
black ink. Take care when removing the needle not
to move the membrane.
4. After 2-5 minutes carefully peel the membrane off
of the plate.
5. Store the plate at 4 °C. It may be desirable to first
incubate the plate for a few hours to regenerate
visible colonies.
6. 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. Then blot the membrane briefly on dry
3MM filter paper.
7. Repeat step 6.
8. Rinse briefly in 2 x SSC, 0.2% SDS to remove
cellular debris.
9. Air dry 30 minutes, bake 1 hour at 80 °C.
10. Hybridize (see Section 4).
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