I had to digest my insert containing plasmid and another vector into which my insert has to be ligated, each with BamHI and NotI.
Upon digestion, I ran the gel to check for results. The digestion seemed to be ok the first time with reasonably good bands. Since I needed more concentration I ran the gel with the same reaction setup (30microL) for the second time.
Well 1 - 100bp marker
Well 2 - Insert in a vector (820 ng/microL, 3microL)
Well 3- Plasmid vector into which the insert has to be ligated (1000 ng/microL, 2microL)
The marker bands were faint while there were absolutely no bands in the other wells. What could be the reason for failure during the second run? I've performed the second time digestion in a span of 40 min after the first one.