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
Since nonspecific nicks within the target sequence increase the background and reduce the resolution of the sequence, it is advisable to use 1-m-long gels or more. The separations on such gels will lead to a greater distance between the bands so that the background, due to the nonspecific degradation products or hybridization-mismatches, will be diluted. For the gel system described in this book an electrophoresis of about 11 hours is optimal for a maximal resolution of the DNA sequence. For an electrophoresis of 11 hours or more a careful choice of the ionic strength of the buffer is required.
Dissolve pellets in 100 ILl of 1 M piperidine (100 ILl piperidine and 900 ILl of water; freshly prepared). > Incubate in a water bath at 90-95 C for 30 0 minutes. 001 TORR). > Dissolve pellet in 100 ILl of water, freeze and lyophilize. > Repeat last step at least twice and dissolve the pellets in 20 ILl of water, divide into two aliquots of 10 ILl each. Samples can now be stored at -800 C until required. > Lyophilize one aliquot and dissolve it into 2 ILl of water, add 5 ILl of sample dye (see 'Materials and Buffers'; if possible dissolve the pellet in 1 ILl of water and 3 ILl of sample dye), heat for 1-2 minutes at 95 0 C, chill in ice/water and load the samples onto the gel.
The buffer is 1 X TBE. > Use a needle and a syringe filled with buffer and remove the air bubbles on the bottom of the gel. The preelectrophoresis is performed overnight with a constant current of 11 rnA (approximately 300V). > Change the buffer in the upper and lower chamber. > Heat up the gel for about 1 hour at constant current (50 rnA). The surface of the gel will reach approximately 45° C. > Switch off, disconnect the bridge (put it into the upper buffer chamber to ensure it does not dry out) and clean the slots with a stream of 1 X TBE, using a synnge.
A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)