single stranded DNA prep

Keith Rand Keith.Rand at molsci.csiro.au
Wed Jun 9 19:43:05 EST 1999


In article <7jk9p6$nso$1 at mozo.cc.purdue.edu>, mhari at expert.cc.purdue.edu
(Malathi Hari) wrote:

>  I am trying to prepare single stranded DNA from a construct in pBS for
> site directed mutagenesis. After a few tries I am still getting a lot of of
> ds plasmid DNA in my prep, as deduced by the ability of the ssDNA to transform
> bacteria and digestion of the DNA;eading to fewr number of transformants.
> I was wondering if anybody out there would have soem ideas.
> In brief this is what I do :
> Grow up an o/n
> Inoculate 1:100 and grow up for 1 hour (I have tried 1.5 ml and 15 ml volumes)
> Add R408 rescue phage ( I don't exactly know the pfu, but I do get lysis as
> seen by the appearance of coagulum)
> Spin down and to sup add PEG/NaCl and keep on ice 30 min to 2hrs
> Spin at high speed
> Dissolve pellet in TE
> Phenol extract
> Chloroform extract
> Ethanol ppt
> 70% wash
> 
> ANY IDEAS???
> 
> Malathi

It sounds likely that the contaminating ds dna is due to the lysis that
you have observed. At least in theory, infection by the M13 helper
shouldn't cause lysis. Perhaps the E.coli strain that you are using does
tend to lyse when infected with M13, so a switch of coli strain would
help. Otherwise, any chance of contamination with other types of phage?
Could it that your construct is producing something that causes lysis? It
is even possible that increased transcription of your insert may occur
after M13 infection. Have you run pBS alone as a control, as this would
help narrow down the problem.

-- 
Keith Rand,  Sydney Australia



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