By Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)
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Additional info for A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA
The teeth of the comb (B) should be 20 to 25 mm long and have a tight fit. The distance between two teeth is about 3 mm. 52 SEPARATION OF REACTION PRODUCTS Fig. 6. Setting up the gel for electrophoresis To avoid an overheating of the upper part of the gel, the glass plates should not be in direct contact with the upper buffer chamber. Therefore a space of about 2 cm is maintained between the glass plates and the upper buffer tank. The gel is loaded with the samples and Whatman 3 paper bridge (1) is placed between the buffer tank and the gel.
The Restriction Digest We found that the best results were obtained when diluted genomic DNA was digested overnight in small aliquots. The incubation buffers were always prepared according to the suppliers recommendations with the following modification: we did not use bovine serum albumin in order to avoid any possible DNAse contamination that may increase the number of nonspecific nicks within the DNA. The sterile-filtered and ten-fold concentrated incubation buffers were stored frozen in aliquots.
7) Complete the transfer system by carefully placing 16 sheets, one after the other, of Whatman 17 paper (also soaked with 1 X TBE as described above) onto the membrane (papers should be just wet but not dripping with buffer). Note: As for the classical Southern-blots the upper layers offilter paper on top of the nylon membrane should never come into contact with the lower layer offilter paper. (8) The anode electrode is placed on top of the second Whatman layer; place a weight of 2 kg on top of it (bottle(s) with water will do the trick).
A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)