Problem isolating M13 ssDNA

Stephen Klautky agoodrid at vaxa.weeg.uiowa.edu
Mon Aug 24 01:29:00 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



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