Cutting M13 dsDNA

Ted Michelini tedm at darkwing.uoregon.edu
Sat Nov 4 01:07:35 EST 1995


In article <gareth_p.8.30955A00 at vcp.monash.edu.au>,
gareth_p at vcp.monash.edu.au (Jo Caine) wrote:

> I have just completed a mutagenesis experiment using M13 vector 
> and I'm having trouble cutting the dsDNA of the M13 with the Nde/Bam.
> I have isolated the dsDNA with a homemade resin based miniprep.  
> Does anyone have any suggestions of how to get cuttable dsDNA?
> Do I have to go to CsCl preps?
> 
> Thanks in advance.
> 
> Jo Caine
> Victorian College of Pharmacy
> Melbourne
> Australia


Jo,
   We always had to wash the infected E. coli cell pellet MANY times to
minimize the amount of ssM13 in our dsM13 preps, remember the dsM13 is
just an intermediate in the rolling circle replication of the phage and as
such is present in a much smaller amount. I belive we still always had
some ssM13 present. I guess hydroxy appetite could give you what you want,
its easier than CsCl good luck

regards 
Ted Michelini
Institute of Molecular Biology
University of Oregon



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