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Additional info for A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA
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. Control Samples: Take 1-2 ILl of specific reaction product per control sequencing lane and add 25 JLg E. coli DNA (in water). Dry the sample in the speed vac and dissolve pellet in water and sample dye as described above for genomic DNA.
Cut out the pieces of the gel one after the other using a scalpel blade. If the gel remaining on the glass plate has also to be transferred take care that it does not dry out; cover the cut side with a small strip of Saran wrap. Preparation of the Electrophoretic Transfer System: > Cut a sheet of Gene Screen to the exact size of the gel to be transferred (wear gloves to handle the nylon membrane); cut one comer to aid subsequent orientation of the Gene Screen membrane. > Float the membrane on 1 X TBE at room temperature for at least 10 minutes.
5-methyl cytosine can be distinguished from cytosine by its lack of reaction with hydrazine resulting in the disappearance of a band in the C-specific sequencing lane. To ascertain that the absence of a band in the sequence represents 5-methyl cytosine and not thymine (arising by a deamination of 5-mC), thymine-specific sequencing reactions have to be performed. , 1984). The most convenient procedure, based on potassium permanganate oxidation of thymidine, was described by Rubin and Schmid (1980).