magic mp & sequenaseDIR

Bruce Roe broe at aardvark.ucs.uoknor.edu
Fri Dec 4 07:48:00 EST 1992


In article <1992Dec3.110653.65 at ntet.nctr.fda.gov>, wmelchior at ntet.nctr.fda.gov writes...
	<stuff deleted>
> 
>Why wouldn't nicked templates cause noise by leading to premature 
>termination (at nicks) of synthesis from primers?  I've been under the 
>impression that avoiding this type of error was one of the advantages of 
>dye-terminator chemistry for fluorescence sequencing:  You see only 
>correctly terminated chains.
> 
>Responding directly to Bruce's suggestion:  The first time I used silica 
>purification (sevral years ago), I got miserable sequences, which I
>attributed to the nicking that takes place during this procedure.  But the
>revised protocol I alluded to in my earlier message gives good results, so
>either the newer method doesn't cause as much nicking or that wasn't really
>the problem.  In either case, it's not necessary to avoid this type of 
>purification just because you're doing ds sequencing.  (The silica method 
>gives me good results both with 35S and with dye-terminators.)
> 
>WMELCHIOR at NTET.NCTR.FDA.GOV

Bill, et.al
	In our hands, preps that have high backgrounds with radioactive incorp.
or the dye-terminators, due we think to nicking or shearing, seem to have
lower backgrounds when radioactive or fluorescent labeled primers are used.
I can only assume that the nicks are soooo random that stops at the nicks
get blurred in the background.  However, badly prepared templates give
lots of noise via ether labeling method, but more noise with incorporation.
A second thought is that the presence of sheared genomic DNA contamination
also gives increased background, but have no idea which (nicked template
or genomic contamination) is the major contributing factor to the bkg.
It also seems that the silica-based methods, including Magic MiniPreps and
Prep-a-Gene, bind the relaxed (nicked dsDNA) and linear (genomic DNA) which
is not released under the conditions that release the supercoiled plasmid,
thus cleaning up the preps.
That's my 2cents worth.........bruce
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
 \  Bruce A. Roe               Professor of Chemistry and Biochemistry /
 /  University of Oklahoma     INTERNET: BROE at aardvark.ucs.uoknor.edu  \
  - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -  - - -



More information about the Methods mailing list