Bio-Rad C/P Lift Membranes - Manual (Page 6)

2. Cut one corner of the plastic bag and pipet
prehybridization solution in:
1 mM EDTA
0.5 M NaHPO
4
pH 7.2 *
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.
6 7
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
4
pH 7.2
5% SDS
2. Wash membrane at 65 °C, 2 times, for 30-60
minutes each in the following:
1 mM EDTA
40 mM NaHPO
4
pH 7.2
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 NaHPO
4
pH ≈ 7.2 MW g/l
1 M Na
2
HPO
4
. 7H
2
O 268.07 134
Add 4 ml 85 % H
3
PO
4
[ 1 M in Na + ]
LIT267B 10/28/98 10:12 AM Page 6