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
Under appropriate conditions (variety of buffers, heat treatment, IN treatment or a combination of both) it was possible to bind nearly 100 % of the input DNA onto the different membranes. However, as we shall see, such a strong binding of DNA to the membranes is undesirable for molecular hybridization. The DNA binding capacity of a filter was determined as follows: total genomic DNA was labeled radioactively by nick-translation and then was submitted to one of the sequencing reactions of Maxam and Gilbert.
STEP-By-STEP PROCEDURE 63 (5/4) Place the gel, covered with the Saran wrap, on top of the filter papers (avoid air bubbles); to help visualize the bubbles use a spotlight placed at a low angle to the gel. Press the gel gently onto the Whatman layer to get rid of air bubbles by using a piece of soft paper. Peel off the Saran wrap carefully. Place along both sides of the gel spacers of the same thickness as for gel electrophoresis so that the upper and lower layer of Whatman papers are not in direct contact.
However, one should bear in mind that irrespective of the size of the genome, always the same quantity of DNA has to be taken (number of cuts per 1000 nucleotides) for each chemical sequencing reaction. 5. Example ora highly resolved genomic sequence A 1-m long sequencing gel was cut into three pieces, transferred to nylon membranes and hybridized with a 120-nucleotide-Iong single-stranded DNA probe. The lower part of the gel (righthand panel) gave a resolution between nucleotide position@ and @from the end of the restriction fragment, the middle part (middle panel) between nucleotide position@and @) and the upper part (left-hand panel) between nucleotide position @ from the end of the restriction fragment and the exclusion size of this 8 % gel which is approximately 300 nucleotides.
A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)