ssDNA and SDM

lhyatt at hkucc.hku.hk lhyatt at hkucc.hku.hk
Thu Dec 9 04:01:55 EST 1993


	Hi!

	I have a couple of questions about site directed mutagenesis, and
preparation of single stranded DNA.

1)	I am trying to make ssDNA of a KS clone containing a 5KB insert. I am
using the Stratagene helper phage VCSM13, and KL-1 Blue cells. The first few
times I did this prep on a small scale (1.2ml), I got nice clean ssDNA. I
scaled up to 10ml, and got nice clean DNA the first couple of times. I used the
last of the 50ml phage stock (about 2-3 weeks at 4C), and got smears on the gel
instead of a nice clean band. I grew up more cell culture from the original
plate, infected with helper phage, and repeated the ssDNA prep. Now I don't
even get a smear around the right size, but a band corresponding to the helper
phage DNA and a much larger band(>23kb). I am about to re-transform XL-1 Blue
cells with my dsDNA, and repeat the process, in case there is a problem with my
plasmid.
	Has anyone else out there used this method (or another), and could give
me some advice. I am getting very frustrated, as I seemed so close to getting
my mutant, ran out of DNA, and now can't make any more. (I have repeated the
ssDNA preps using 1.2ml and 10ml scale, using heat or phenol to deproteinise,
adding RNase and/or DNase to the phage stock or neither, using PEG/NaCl or
PEG/Ammonium acetate to ppt the phage, and compared the different results. So
far I haven't hit the jackpot.)

2)	My second question (yes, that was just one question!) concerns site
directed mutagenesis using streptavidin coated magnetic beads. The protocol
says to cut with the first restriction enzyme, endfill using dCTP,dGTP,dATP and
biotin dUTP, cut with the second enzyme, and add this mix to the streptavidin
coated beads. Initially, I got a reasonable amount of binding (original mix vs.
supernatant from beads on an agarose gel), but there was a problem somewhere
else. Now I do not seem to get any binding. Is there any way of finding out if
the biotinylation step has worked? Do these beads lose their streptavidin
coating? Has anyone out there used this method to do SDM, and do you have any
suggestions/clever tricks to get it to work?

	Well, those are my questions. I hope there is someone who can help me,
as I would really like to get my mutation soon. This was supposed to be the
start of a 36 month project, and I am already 33 months through!! (not doing
SDM all that time)

	Thanks for taking the time to read this, and for answering (PLEASE).

	Lucy

LHYATT at HKUCC.HKU.HK



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