What's the best mini-prep?

[C.J. Hussussian, MD]

>i agree here more or less.  if you're looking for the cleanest DNA around,
>my experience is that Qiagen preps provide it.  however, i have also found
>that the average Qiagen prep gives enough DNA for about 1/2-1
>autosequencing reaction (5 ml of cells usually gives me about 500-1200ng of
>DNA) which is a waste of time and money.  your results may vary of course. 
>you dont' say what sequencing protocol you will be using, but for automated
>sequencing with the ABI 373A (disclaim: no connection, just run the
>sequencer in this lab) we have developed a protocol which uses Promega
>(disclaim once again, no connection, etc.) Wizard Mini-preps followed by
>EtOH ppt.  it's relatively quick and painless and not "too" expensive.


I agree that Quaigen preps provide for very clean DNA (we can't
distinguish it from CsCl banded DNA). However, we routinely get much
higher yields than stated above. We usually get 20-30 ug of DNA per 5
ml prep (using Quiaprep mini-columns). The yield will vary considerably
depending on the host as nucleases will quickly degrade plasmid DNA and
the proteoglycans can quickly overload the columns. We had low yields
several months ago trying to use the Invitrogen One-Shot cells with the
columns. These problems vanished when we switched to XL1-Blue or
DH5-alpha.


C.J. Hussussian
NIH/NCHGR
49/4B83
Bethesda, MD 20892
(301)402-2029
cjh at helix.nih.gov