M13 DNA prep kits?

Em ekhatipoNO at NOmidway.uchicago.edu
Tue Sep 18 11:48:30 EST 2001


Hi,
Could someone tell me if there is a cheeper alternative to Quiagen Qiaprep
96 M13 kit? I would also appreciate your comments on alternative (no-kit)
cost efficient methods of M13 ssDNA isolation could be used for multiple
preps (50+ samples at a time) that would result in DNA pure enough for
sequencing (fluorescent dye termination method, gel or capillary machines).
I am aware of the method utilizing NaI precipitation of ssDNA (resusp. phage
pellet in 2MNaI/Tris/EDTA buffer, add 2.5 ml EtOH and spin within 10 min).
The method (in 1.5ml test tube format) gave me pure enough DNA for
sequencing in the past (on a gel slab ABI machine). It seems it can be
modified to fit 96-well format, but there is a couple of concerns I would
like you to comment on. NaI/EtOH is believed to preferentially precipitate
ssDNA only during first 10 min after EtOH addition. Longer time will result
in contamination with dsDNA and (?) proteins. Spinning 96 plates is usually
carried out at lower speeds then test tubes. That means - increased times of
spinning => contamination with dsDNA/proteins. Modern capillary sequencers
are much more sensitive to contamination with proteins than coventional gel
electrophoresis-based methods.

I would appreciate to hear an advice from someone who dealt with similar
problems in the past.

Thank you.
Emir Khatipov
Univ. Chicago





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