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3.
Replace the depurinating solution with denaturing solution. Denaturation for at
least 30 minutes is recommended for most applications with a change of solution
after 15 minutes.
4.
Replace denaturing solution with neutralising solution and gently shake the gel
for least 30 minutes, replacing with fresh solution after 15 minutes.
5.
The DNA is then transferred to a Hybridisation membrane cut to the size of the gel.
At this stage, a corner of the membrane should be cut to assist later in orientation.
Transfer may be carried out by capillary blotting, or more efficiently by vacuum
blotting. Vacuum blotting offers significant advantages over capillary blotting,
namely: a)
Transfer is completed in 1 hour, instead of at least 4 hours for capillary
blotting, or more usually overnight.
b)
Superior results are obtained with vacuum blotting. Because of the rapid
transfer time, there is less lateral diffusion of the DNA during transfer to the
Hybridisation membrane. This results in sharper bands on autoradiographs.
Excellent blots may be prepared, quickly and conveniently, using the Thermo
Vacu-Aid apparatus.
Capillary Blotting
1.
Capillary blotting requires a buffer reservoir containing 10-20 x SSPE (SSC) (e.g. a
bioassay dish) with a raised support for the gel (e.g. an inverted sandwich box).
Two sheets of Whatman 3MM filter paper cut to the same width and slightly
longer than the gel should be pre-wet in 10-20 SSPE (SSC) and placed across the
raised support so that the ends are submerged in the buffer reservoir. This serves
as a wick for the flow of the buffer through the gel (see Figure 2.2: Capillary Blot
Assembly).
2.
Invert the gel and place centrally on the filter paper. Any filter paper exposed at the
edge of the gel should be covered in Saran Wrap, used X-ray film or similar, to
prevent flow of buffer around the edge of the gel.
© Thermo Scientific, May 2003. Issue 7
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