s_laub, on Sep 7 2009, 02:23 AM, said:
I just started working in a lab, but I've always been using 300 ul of 70% EtOH, followed by 300 ul of 99% EtOH after a miniprep and my supervisor said that's fine. But the preps are obviously dirty because they can't be cut with REs. So should I always wash with a minimum of 1 ml?
As a few others have stated, the actually volume isn't the most critical issue, so I wouldn't stress too much over it; you PI's advice is probably the one to follow.
Having said t hat, when we had digestion issues, we did one of two things. Once the DAN was resuspended, we put it through a second EtOH precipitation (or did a PCR cleanup on it). This is fine for most contaminants.
If, however, the repeat precipitation didn't do the trick, we went back and recultured, but we used LB. A few of our group found that overnight culturing with the really rich media gave higher yields of cells and plasmids (no surprise there!), but the DNA quality was lower. Growing cells in LB gave lower yields (fixed by larger cultures!) but better digestion.
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