Problem isolating M13 ssDNA

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Mon Aug 24 01:39:24 EST 1992


>Hi,
>
>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.
>
>I follow the procedure for ssDNA isolation in Current Protocols.
>
>When the phage is precipitated I see a phage pellet - offwhite
>in color.  I try to remove as much of the PEG as possible.
>I understand that contaminating PEG can interfere with DNA
>polymerase rxns but can it interfere with extraction efficiency?
>
>After resuspension of the pellet in TE I extract with 1/2 vol.
>of phenol and spin in a microfuge.  There is a small pellet at
>the bottom of the tube after this step which varies in size
>from tube to tube.  Is this a problem?  I have seen this before
>and still obtained ssDNA from the preps.
>
>After EtOH ppt of the extracted supernatant and resuspension there
>are still some fine particles floating about which are resistant to
>resuspension.  I heat the samples at 50 degC but they don't go into
>soln.
>
>When I run the gel I see no/little signal in the region where I
>expect my ssDNA (I run a previously purfied DNA+insert and M13
>ssDNA as controls).  In addition I usually see a band of weak but
>distinct intensity running just above the top band of the 1 kb Ladder.
>I expect this is contaminating genomic DNA.
>
>So, are there any hints one can give me on obtaining good yields
>of ssDNA from M13.
>
>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


The PEG can be a real problem.  I always aspirate after the first spin,
then respin (20 sec) and it is amazing how much peg is stuck to the walls. 
If you reaspirate this it might help.

Hope this helps.

Klaus
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Don't try and fix it if it ain't broke".
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