Agarose gel electrophoresis of PCR products
A 400 ml gel (1% agarose in 45 mM Tris Borate–1 mM EDTA buffer (pH 8.3) containing 10
µg/ml 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. 4 µl of 6X loading buffer (0.25% bromophenol blue, 0.25% xylene
cyanol and 40% sucrose in sdH20) was added to the entire PCR reaction and mixed. An 8.5 µl
aliquot was removed and loaded into a well of the 1% agarose gel supported in a BioRad SubCell Model 192 horizontal gel system. Gels were run for 15 hr at 35 VDC or 6 hr at 80 VDC.
The DNA bands were visualized using a Fotodyne UV trans-illuminator and the results were
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. Using 1% agarose gels
allowed routine screening of insert sizes. However, for the larger ORFs (>1500 bp) that were
being cloned, the PCR products for ORFs were re-run on a 0.7% agarose gel and scored.