Bio-Rad Trans-Blot® SD Semi-Dry Transfer Cell - Manual (Page 8)

b. Power conditions during transfer may have changed. It is important to have constant
current during the course of the run. If the buffer is less concentrated than 0.5x, high-
er voltage will be required to maintain the recommended current. If the voltage limit
is not set high enough, the current will drop below the optimum range during the run,
thereby reducing DNA/RNA migration. Readjust the power supply parameters.
c. Optimum transfers of plasmid, vector, and PCR DNA are achieved when blotting
the gel at a constant current of 3.55 mA/cm 2 for the recommended time of 10 minutes.
Optimum transfers of RNA are achieved when blotting the gel at a constant current
of 3 mA/cm
2 for 30-35 minutes. More difficult transfers (i.e. genomic DNA) may
require slightly longer transfer conditions at lower current.
2. Poorly blotted or diffused transfer.
a. There may be a poor contact between the agarose gel and the transfer membrane.
Roll out the gel with a pipette prior to transfer to get rid of air or buffer bubbles. Also
be sure to remove air and buffer bubbles from the blot paper.
b. The gel may be too thin, causing uneven electrical contact between the gel stack and
the electrodes. The gel must be 6 mm thick, and Bio-Rad's ExtraThick Blot Paper
must be used with the kit to insure proper electrical contact.
c. The gel may be too hot and is partially melting. See Section A 1-a of troubleshooting.
d. Very small fragments of DNA tend to diffuse during electrophoresis and blotting
even if run in high percent agarose gels. Resolution is also not necessarily improved
with high percentage agarose gels.
e. The transfer membrane being used may not properly bind DNA or RNA. Try using
a control membrane, or a different lot, or a different brand of membrane.
B. Poor Detection Sensitivity
1. Poor DNA probe labeling.
a. Not enough signal is hybridized to the target DNA for detection. Labeled DNA probe
may not be properly labeled. Check labeling reaction controls to be sure correct tem-
plate DNA is being used and that reaction is working properly.
b. Target DNA may not have completely transferred to the membrane. See Section A of
troubleshooting. Check agarose gel for presence of DNA following transfer to deter-
mine whether transfer occurred or not.
c. Specific activity of the probe may not be high enough for standard detection condi-
tions. Determine specific activity and total cpm of probe added during hybridization.
d. Hybridization conditions may be too stringent. Alter the hybridization conditions to
reduce stringency. For more specific recommendations, refer to the Zeta-Probe GT
instruction manual.
C. High Background
1. Increase stringency of hybridization conditions to reduce non-specific probe binding.
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