Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)'s A Laboratory Guide to Genomic Sequencing: The Direct PDF

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

Sample text

Add 750 JLl of precooled ethanol, mix thoroughly by inversion, chill at -700 C and centrifuge as described above. > To wash the pellet, add carefully 1 ml of 70 % ethanol/water, centrifuge for 5 minutes and carefully pour out the supernatant and centrifuge again for 1 minute. > Remove the residual ethanol with a drawn out glass capillary and dry the pellet in the speed vac. > The DNA is ready for piperidine treatment (see at the end of the chapter). LI of water by tapping the tube. LI of formic acid

The number of cuts per target DNA molecule can greatly affect the resolution of the genomic sequence. If at least one of the two cuts is located within the probe region, for example, two or three hybridizable fragments are created. Such fragments result in additional unspecific bands within the sequencing lanes and makes the sequence more difficult if not impossible to interpret (see 'Trouble-Shooting Guide': Fig. 2112). --J<-.. 4. T-specific reaction Comparison ofG- and T -specific reactions of potentialmethylation sites (b, c, d) in the upstream area (upper strand) of the avian vitellogenin gene II (L = liver DNA, E = erythrocyte DNA.

The genomic DNA was digested overnight at the temperature recommended by the supplier and with a three-fold excess (units/JLg DNA) of restriction enzymes (6 basepair cutters) and a ten-fold excess (units/JLg DNA) for 4 base-pair cutters. Following digestion, the genomic DNA should be treated at least once by phenol extraction. After the digestion with restriction enzymes it is routine to perform at least one classical Southern-blot analysis (Southern, 1975) to check that 1) the DNA was digested to completion and 2) that there is only one DNA frag- INTRODUCTION 33 ment, hybridizing with the radioactively labeled probe.

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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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