80
FENG QIAN AND GREGORY G. GERMINO
If the the blot will be probed for transcripts around 2 kb or smaller, rinse
the gel in two changes of 20 x SSC to remove the formaldehyde. The gel is
then placed in contact with the nitrocellulose filter or nylon membrane and
the RNA is by capillary transferred to the membranes actioninan ascending
flow using 10 x SSC as the transfer buffer. Furtherdetails of this procedure
can be found in the section describing Southern blots as the final set-up is
identical.
Recently, a number of transfer devices have become commercially available which greatly decreases transfer times from 12 h to less than 1 h. The
majority of transfer apparatuses use a negative pressure (vacuum) to draw
the nucleic acids out of the gel onto the membrane. We, however, have
excellent results using the Posißlot® pressure blotter from Stratagene.
This system uses a positive pressure to drive RNA (or DNA) molecules
from the gel onto the membrane. Transfer is completed within 1 h and
the efficiency of transfer is similar to that obtained with standard capillary
technique. After blotting, the membrane is UV -crosslinked or baked at 80° C
for 2 h.
Northern The conditions for prehybridization, hybridization and washing of RNA
hybridization immobilized on membranes are essentially the same as those used for
DNA. We use the buffer described by Church and Gilbert (CG buffer).
1. Prehybridize the membranes for at least 2 h in CG buffer at the appropriate temperature.
- 1 x CG buffer
- 0.5 M Na2HP04 (pH 7.2}
- 1 o/o BSA
- 7% SDS
- 1 mM EDTA
2. Add the denatured radiolabeled probe directly to the prehybridization
solution. To detect low-abundance mRNAs, use at least 0.1 1-1g of probe
whose specific activity exceeds 2 x 10 8 cpm/!J.g. Continue incubation for
16-24 h at the appropriate temperature.
3. Wash the membrane for 20 min at room temperaturein 1 x SSC, 0.1%
SDS, followed by 2-3 washes of 15 min each at 50- 65°C in 0.2 x SSC,
0.1% SDS.
4. Expose the membrane to X-ray film (Kodak XAR-2 or equivalent) at
- 80°C with an intensifying screen.
FENG QIAN AND GREGORY G. GERMINO
If the the blot will be probed for transcripts around 2 kb or smaller, rinse
the gel in two changes of 20 x SSC to remove the formaldehyde. The gel is
then placed in contact with the nitrocellulose filter or nylon membrane and
the RNA is by capillary transferred to the membranes actioninan ascending
flow using 10 x SSC as the transfer buffer. Furtherdetails of this procedure
can be found in the section describing Southern blots as the final set-up is
identical.
Recently, a number of transfer devices have become commercially available which greatly decreases transfer times from 12 h to less than 1 h. The
majority of transfer apparatuses use a negative pressure (vacuum) to draw
the nucleic acids out of the gel onto the membrane. We, however, have
excellent results using the Posißlot® pressure blotter from Stratagene.
This system uses a positive pressure to drive RNA (or DNA) molecules
from the gel onto the membrane. Transfer is completed within 1 h and
the efficiency of transfer is similar to that obtained with standard capillary
technique. After blotting, the membrane is UV -crosslinked or baked at 80° C
for 2 h.
Northern The conditions for prehybridization, hybridization and washing of RNA
hybridization immobilized on membranes are essentially the same as those used for
DNA. We use the buffer described by Church and Gilbert (CG buffer).
1. Prehybridize the membranes for at least 2 h in CG buffer at the appropriate temperature.
- 1 x CG buffer
- 0.5 M Na2HP04 (pH 7.2}
- 1 o/o BSA
- 7% SDS
- 1 mM EDTA
2. Add the denatured radiolabeled probe directly to the prehybridization
solution. To detect low-abundance mRNAs, use at least 0.1 1-1g of probe
whose specific activity exceeds 2 x 10 8 cpm/!J.g. Continue incubation for
16-24 h at the appropriate temperature.
3. Wash the membrane for 20 min at room temperaturein 1 x SSC, 0.1%
SDS, followed by 2-3 washes of 15 min each at 50- 65°C in 0.2 x SSC,
0.1% SDS.
4. Expose the membrane to X-ray film (Kodak XAR-2 or equivalent) at
- 80°C with an intensifying screen.