Bio-Rad Zeta-Probe Membranes - Manual (Page 14)

Problem
3. High background observed
throughout membrane on autora-
diograph
Solution
The major contributors to background are
unincorporated label, small radioactive decay
products, and small probe fragments result-
ing from nick translation or random priming.
1. Use a desalting gel column to remove
unincorporated label. Bromophenol Blue
is a useful indicator. The peak of unin-
corporated label overlaps with, and
slightly precedes the Bromophenol Blue
in a desalting column.
2. Use the probe as soon as possible after
preparation, since decay results in frag-
mentation.
3. Reduce exposure of the probe to DNase
I during nick translation to increase
average probe length.
4. Use a different heterologous nucleic acid
in the prehybridization and hybridization
mixtures. Sonicate it thoroughly and
denature it before use.
5. For RNA probe, post-hybridization
washes: remove SDS by washing 3x in
100 mM sodium phosphate (pH 7.2),
then wash with 10 µg/ml RNase A,
0.3 M NaCl, 10 mM Tris-HCl, pH 7.5,
1 mM Na
2
EDTA at 37°C for 15 min.
23
Section 7
Zeta-Probe Membrane
Troubleshooting Guide
7.1 Nucleic Acids Problem
22
Nucleic Acids (Continued)
Problem
1. Fragments greater than ~1,000 bp
cannot be electrophoretically
transferred from polyacrylamide
gels after base denaturation, even
at increased volts/hours
2. Very large fragments cannot be
electrophoretically eluted from
agarose gels
Solution
We have observed that fragments
>~1,000 bp can become trapped in
polyacrylamide gels if they are base dena-
tured and neutralized after electrophoresis,
whereas nondenatured fragments will
transfer completely up to at least 2,000 bp.
Solve this problem in one of three ways:
1. Omit pretreatment, and transfer ds
DNA. Alkaline fix post-blotting.
2. Run gel electrophoresis under
denaturing conditions, and omit base
denaturation step and neutralization
step prior to transfer.
3. Omit base denaturation step, and dena-
ture gel instead with 1 M glyoxal, in
25 mM sodium phosphate, pH 6.5,
50% DMSO for 1 hr at 50°C. Then
transfer directly.
1. Solutions 1 and 2 to problem #1 can
also be applied to agarose gels.
2. Depurinate prior to transfer by soaking
the gel in 0.25 M HCl for 20 min, 6 then
soak the gel in transfer buffer for
10–15 min.
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