By Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)
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Additional resources for A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA
Mix by tapping the tube and chill on ice. > Add 30 ILl of hydrazine. > Mix by tapping the tube and centrifuge a few seconds in a microfuge. > Incubate sample at SW o C for 10 minutes. > Add consecutively 200 ILl of hydrazine stop buffer and 750 ILl of cold ethanol. > Continue as described for G-reaction (* * *). T -reaction: > Dry 50-75 ILg of digested genomic DNA in the speed vac. > Dissolve pellet in 5 ILl of water. > Denature DNA at 90 C for 2 minutes; then quick 0 chill in ice/water. 27x 10- 4 M potassium permangnate (KMn04) .
Centrifuge tubes for 20 minutes in a SS-34 Sorvall rotor at 15 000 rpm at 0 0 C. > Pour out the supernatant very carefully and centrifuge again for a few minutes in the microfuge. > Remove the residual ethanol with a drawn out glass capillary. 5 mM). > 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.
52 SEPARATION OF REACTION PRODUCTS Fig. 6. Setting up the gel for electrophoresis To avoid an overheating of the upper part of the gel, the glass plates should not be in direct contact with the upper buffer chamber. Therefore a space of about 2 cm is maintained between the glass plates and the upper buffer tank. The gel is loaded with the samples and Whatman 3 paper bridge (1) is placed between the buffer tank and the gel. 5 cm above the top of the glass plates. The wells (2) have to be cleaned before loading the gel.
A Laboratory Guide to Genomic Sequencing: The Direct Sequencing of Native Uncloned DNA by Dr. Hanspeter Saluz, Dr. Jean-Pierre Jost (auth.)