A Laboratory Guide to Genomic Sequencing: The Direct - download pdf or read online

By Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)

ISBN-10: 3034893027

ISBN-13: 9783034893022

ISBN-10: 3764319259

ISBN-13: 9783764319250

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Read Online or Download A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA PDF

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Additional info for A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA

Example text

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.

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A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)


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