Bio-Rad Zeta-Probe Membranes - Manual (Page 15)

Problem
7. No autoradiograph signal
Solution
1. After transfer, stain the gel to check that
transfer was complete. If not, increase
transfer time and/or voltage of transfer,
or see solution to problem #1 above.
2. Be sure probe is denatured by boiling or
heating to 65°C for 5 min in 50%
formamide prior to hybridization.
25
Problem
4. Localized high background
observed on autoradiograph
5. Lane background or extra bands
6. Low autoradiograph signal
Solution
1. Make sure the membrane is free-floating
within the plastic bag during hybridiza-
tion. Membrane/bag contact during
hybridization can cause background.
Add more hybridization solution.
2. Make sure not to pinch the membrane
when sealing the plastic bag prior to
hybridization.
3. Be sure no bubbles exist in the
hybridization bag.
1. Indicates contaminated template. Make
sure the probe is synthesized with the
pure template of choice.
This problem may occur when total genomic
DNA is probed for single-copy or low copy
number genes.
1. Incorporate 10% dextran sulfate in the
hybridization mixture. This polymer effec-
tively reduces the solvent volume, there-
by increasing the concentration of the
solutes and enhancing hybridization.
Refer to Section 4.3.
2. Increase exposure time to increase sig-
nal-to-noise ratio.
3. Increase sample load on the gel.
4. If low signal is accompanied by low
background, probe concentration can
be increased 2- to 4-fold.
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Nucleic Acids (Continued)
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