CsCl preps for ABI sequencing reactions

Anthony Otsuka ajotsuka at rs6000.cmp.ilstu.edu
Tue Mar 21 09:58:45 EST 1995


I am also curious about the use of CsCl-purified DNA for Sanger
sequencing.  I have had two graduate students prepare CsCl-
purified DNA by the protocols in Maniatis.  The DNA was dialyzed
extensively and used for sequencing.  The bands were fainter
than the equivalent amount of mini-prepped DNA.  This was
curious, since I thought removal of the RNA and the negative
supercoiling of the DNA should improve the efficiency of
primer annealing and DNA sequencing.  Thinking that the problem
was due to a lack of experience, I also made a preparation
of CsCl-purified DNA, and had the same poor result.  I tested
both the supercoiled DNA and nicked DNA bands from the
gradient.  Both worked poorly.  If the CsCl is inhibiting
the reaction, then how much cleaning is necessary?  Do
we need to desalt the DNA over a column?  Ethanol precipitation?
More extensive dialysis (our usual is 250 volumes 4 times --
2h x 3 and overnight 1x)?  Please post your experiences.
My experience is entirely anecdotal and has not been subjected
to controlled experimentation.

Thanks, Anthony Otsuka



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