bernard at elsie.nci.nih.gov
Sat Jul 9 19:59:28 EST 1994
In article <genecutl-080794200752 at kos2mac23.berkeley.edu>, genecutl at mendel.berkeley.edu (gc) writes:
> I've been using a boiling-lysis protocol for mini-preps and it used
> to work beautifully. The protocol involves vortexing the cells in
> STET buffer, boiling, spinning down the precipitated crap, then
> EtOH ppt the sup.
> The pellet I got from this has a huge amount of protein in it, but,
> surprisingly, my restriction digests always looked great and I even
> had much greater success sequencing this DNA than DNA from alkaline
> lysis preps.
> The problem is now the mini-prep gods no longer smile on me and I'm
> lucky to get a single sample to work out of twenty. What can have gone
> wrong? Please don't tell me I have to go back to phenol/chloroform
First, a naiive question - what bacteria are you using? If they are not
EndA -ve then the DNA may get eaten. However, it sounds as if you are getting
no DNA at all instead of degraded material. My favourite protocol is by
Zhou et al. "Mini-Prep in Ten Minutes" Biotechniques 8(2)172-173 (1990).
The resultant DNA is fine for digestion.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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