Plaque Blotting; Southern Blotting - Thermo Scientific Shake n Stack 6240 Operating And Maintenance Manual

Pre-washing is carried out as required by incubating in at least 200ml of prewash
solution (see Appendix I) for at least 30 minutes at 50°C. Longer incubations and
several changes of buffer will assist in pre-washing. When the colonies are
sufficiently broken up, residual colony debris may be removed by gently rubbing
the colonies with a gloved finger.
This procedure is particularly advantageous when screening with oligonucleotides.

Plaque Blotting

The above procedure, with some minor modifications may also be utilised for the
screening of bacteriophage plaques as follows: -
1. Aliquots of the bacteriophage stock should be mixed with plating cells and plated in
soft agarose. Incubate at 37°C until plaques are approximately 0.2-0.5mm in
diameter (approx. 10-12 hours).
2. Chill the plates for about 1 hour to set the top agarose.
3. Place a Hybridisation membrane cut to size (or pre-cut disc) on to the surface of
the agarose and leave for at least 30 seconds. Orientation points should be marked
with a sterile needle. Further replicas may be prepared by leaving the Hybridisation
membrane for progressively longer periods of time on the surface of the agarose.
4. To process membranes further; proceed as from stage number 5 as for 'Colony
Blotting'. Pre-washing the filters (stage 9) should not be necessary.

Southern Blotting

1. Size fractionation of the DNA is carried out by agarose gel electrophoresis (a
suitable range of horizontal gel apparatus is available from Thermo Scientific).
Before transfer to Hybridisation membranes, the DNA in the agarose must be
treated to ensure efficient transfer and to generate single-stranded DNA
suitable for Hybridisation. Gentle agitation of the gel is essential to prevent damage
to the gel during these steps. The shakers in Shake 'n' Stack, Midi Dual 14 and
Maxi 14 are ideal for this purpose.
2. Depurination to break the DNA into smaller fragments suitable for transfer is
recommended for transfer of all DNA fragments large than 10kb and may assist
transfer of smaller fragments. Place the gel in a solution of 0.25M HCl for 10
minutes at room temperature with gentle shaking.
© Thermo Scientific, May 2003. Issue 7
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