Agarose gel electrophoresis of Primary PCR products from Nested

Agarose gel electrophoresis of Primary PCR products
A 400 ml 1% agarose gel in 0.5X Dingman’s buffer with 0.001% ethidium bromide was cast into
a BioRad Model 192 gel caster with two 51-well combs placed so that a two tiered gel with 48
samples and three molecular weight marker control samples could be run per tier. A 3 µl aliquot
of the primary PCR reaction was removed and added to an 8 µl aliquot of 3X loading buffer
(0.125% bromophenol blue, 0.125% xylene cyanol and 20% sucrose in sdH20; usually made by
diluting a 6x stock solution) . The mixture was loaded into a well of the agarose gel. Either 35
VDC or 80 VDC was applied to the system for 15 or 6 h, respectively. The DNA bands were
visualized using a Fotodyne UV trans-illuminator and the data was documented using a Sony
videographic printer model UP-890MD. The gel was scored for the presence or absence of the
expected size fragment for each ORF.