problems with qiagen preps.

C.J. Hussussian, MD cjh at helix.nih.gov
Sat Jul 16 15:23:50 EST 1994


You should also know that vector and host strain are more critically
important than indicated by the Quiagen manual. After successfully
using the old Quiagen p-20 columns to purify DNA for Dye Primer
sequencing on the ABI, we struggled for months to get adequate yeilds
from the Quiawell kits. We ended up changing host cells from Invitrogen
One-Shot to XL1-Blue to DH5alpha. We now get >95% of our preps with
yields >6 micrograms from 2 ml cultures.

We spoke to a Quiagen scientist in Germany and apparently the wells are
quite sensitive to overload from the proteins and other crap that
doesn't get precipitated out. If the levels of these contaminants
reaches a critical threshold, one's yield drops to near zero. Also, for
optimal results, you need to use a high-copy plasmid. We use the PCRII
plasmid from Invitrogen (TA cloning kit) or Bluescript. Both work
equally well.


Good Luck,

Christopher Hussussian, MD
NIH/NCHGR 49/4B83
9000 Rockville Pike
Bethesda, MD 20892
(301)402-2029
cjh at helix.nih.gov



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