Bio-Rad Trans-Blot® SD Semi-Dry Transfer Cell - Manual (Page 7)

7. Place the safety cover onto the unit and plug the Trans-Blot SD cell into the power sup-
ply. Be sure to maintain normal polarity of the electrodes, i.e. red lead to red outlet and
black lead to black outlet.
Caution: Do not reverse the electrode polarity. This will damage the stainless steel
cathode.
8. Turn on the power supply. The power conditions and transfer times will vary depending
on what type and size of DNA/RNA you transfer.
For plasmid or vector DNA and PCR fragments, set the transfer conditions for a constant
3.55 mA/cm2 of gel area for 10 minutes (i.e. a 7.5 x 15 cm gel with an area of 112.5 cm
2
will require 400 mA constant current for 10 minutes). Normally, the voltage will slowly
increase during transfer in order to maintain constant current (i.e., a 7.5 x 15 cm gel run-
ning at a constant 3.55 mA/cm
2 will experience a voltage increase of approximately 5 V
to 10 V during the transfer).
For transferring RNA, set the power supply for 3 mA/cm 2 constant current. Typical trans-
fer time should be 30-35 minutes. During the course of the run, the voltage should be
about 20 V.
During the transfer, observe the voltage for any significant fluctuations. The voltage will
slowly increase during transfer to maintain constant current. If the voltage is lower,
increase the length of the transfer time. If the voltage increases significantly (i.e. greater
than 25 V) the buffer capacity has expired and the run should be stopped. If the run is
not stopped the gel will overheat and eventually melt. If the voltage requirement is sig-
nificantly lower than normal, the buffer may be more concentrated than 0.5x, and, there-
fore, less voltage is required to maintain the specified current. If this is the case, the
recommended transfer can be completed as long as the power supply is adjusted to oper-
ate at the specified current setting. Care must be taken not to overheat the gel or the
Trans-Blot SD cell.
9. Following transfer, turn off the power supply and disconnect the leads. Remove the safe-
ty cover and the cathode electrode. Discard the blot paper and recover the transfer mem-
brane. Rinse the membrane in 2x SSC. The transfer efficiency can be qualitatively
monitored by restaining the gel and checking for any remaining DNA/RNA.
10. To fix the DNA, saturate a piece of blot paper with 0.4 N NaOH. Place the transfer mem-
brane on top of the saturated pad (DNA side up) for 5 minutes. Rinse the membrane
briefly in 2x SSC, and bake for 30 minutes at 80
°C in a vacuum oven.
To fix RNA, dry the membrane it in a 60 °C oven for 2 hours, or dry it overnight at room
temperature. The membrane is now ready for hybridization. Refer to the hybridization
procedure in the Zeta-Probe GT blotting membrane instruction manual.
Section 5
Troubleshooting Guide
A. Poor Electrophoretic Transfer
1. DNA/RNA molecules remain in the agarose gel.
a. If the gel is hot, the buffer may be too concentrated. The gel is carrying too much
current and has begun melting. Buffer concentration must be 0.5x TBE to maintain
proper transfer conditions. Remake the transfer buffer.
4