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C/P Lift®Membranes
Instruction Manual
Catalog Numbers
162-0162
162-0163
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Table of Contents
Section 1 Introduction.............................................1
Section 2 Colony Screening ....................................1
2.1 Direct Lifts................................................1
2.2 Replica Lifts..............................................3
Section 3 Plaque Screening.....................................5
Section 4 Hybridization Protocol...........................5
4.1 Prehybridization........................................5
4.2 Hybridization ............................................6
4.3 Washes......................................................7
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Section 1
Introduction
C/P Lift membranes were developed for colony and
plaque screening. When handling the membranes,
always wear gloves or use forceps. After blotting, do not
allow wet membranes to come in contact with each
other. Contact may result in the transfer of blotted
nucleic acids from one membrane to the other.
Section 2
Colony Screening
2.1 Direct Lifts
1. Plate cells containing recombinant plasmids onto the
appropriate selective agar. Incubate plated cells until
the colonies are 1-2 mm in diameter.
2. Place a circle of C/P Lift membrane onto the surface
of the plate as follows. 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 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).
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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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 µg/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.
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.
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2. Cut one corner of the plastic bag and pipet
prehybridization solution in:
1 mM EDTA
0.5 M NaHPO
pH 7.2 *
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7% SDS
3. Reseal the plastic bag and incubate briefly at 65 °C
for 5 minutes.
4.2 Hybridization
1. Cut one corner of the plastic bag, remove the
prehybridization solution and replace it with same
buffer.
2. Add denatured probe and remove all bubbles before
resealing the bag. Hybridize for 4-24 hours at 65 °C
with agitation.
3. Carefully remove the hybridization solution by
cutting one corner. Remove hybridized membrane
from plastic bag.
Note: At no stage before washing should the membranes
be permitted to dry.
4.3 Washes
1. Wash membrane at 65 °C, 2 times, for 30-60
minutes each in the following:
1 mM EDTA
40 mM NaHPO
5% SDS
2. Wash membrane at 65 °C, 2 times, for 30-60
minutes each in the following:
1 mM EDTA
40 mM NaHPO
1% SDS
3. After washing, the blotted membranes are ready for
autoradiography. Expose moist membranes between
Saran Wrap or enclosed in a sealable plastic bag. Do
not allow a wet membrane to come in contact with
the film, because wet membrane will stick to the
film.
*1 M NaHPO4pH ≈7.2 MW g/l
HPO
1 M Na
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Add 4 ml 85 % H3PO4[ 1 M in Na+]
pH 7.2
4
pH 7.2
4
.
7H2O 268.07 134
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Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT267 Rev B
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