Problem isolating M13 ssDNA

Warren Wakarchuk num208ww at MBDS.NRC.CA
Thu Aug 27 19:47:02 EST 1992


In article <24AUG199200290632 at vaxa.weeg.uiowa.edu>, agoodrid at vaxa.weeg.uiowa.edu (Stephen Klautky) writes:
>Usually, I work with dsDNA but on occasion I make ssDNA to
>sequence troublesome clones.  
>
>Currently, I am having some trouble with my yields.  The problem
>may be at the phage denaturing step but I'm not sure.  I'm using
>DH5-alpha-F' cells, btw.

>>stuff deleted
>
>P.S. the phage sups infect DH5-alpha-F' cells to make plaques on
>YT plates so I don't think that's the problem.
>
>Any info by posting or e-mail is appreciated.
>
>- Steve Klautky -     AGOODRID at VAXA.WEEG.UIOWA.EDU
>
I'd like to offer a couple of explanations and hints about low yields
for ssDNA preps.
1) In my hands DH5-alpha-F' is a low yield producer of ssDNA, I've had
much better luck with MV1190 (or even JM101 in the olden days).  If
you check the titre of phage you'll probably see only 1 E+10 per ml,
which is quite low compared to the 1 E+12 you can get with other
strains.  You may need to work up a 10 ml prep just to get enough for
a couple of reactions!
2) Your "troublesome clones" may be resonsible for your decreased
yields, many clones simply do not produce ssDNA well. Try a DIGE
(direct in gel electrophoresis) to see if your sup's worth working up.
3) The PEG precipitation method works well, but you have to work to
resuspend the pellets before phenol extracting them, other wise you
lose everything.  Let the pellets sit at RT for 5 min. in TE then resuspend
them, they go into solution much better. I use a glass/NaOCl4 method
now which is much faster and gives twice the yields of phenol
extraction (It is not glass milk but glass fibre filter, and I think
it is a great method).
Hope these tidbits are usefull.
*************************************************************************
Dr. Warren Wakarchuk   Prefered email: wakarchu at biologysx.lan.nrc.ca
                       Alternate     : NUM208WW at MBDS.NRC.CA
National Research Council Canada, Protein Structure and Design
Ottawa, Ont., Canada K1A 0R6 ***THESE ARE MY OPINIONS NOT THOSE OF NRC***



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