how to make sigle-stranded DNA of pET

Don Jackson djackson at welchlink.welch.jhu.edu
Fri Nov 25 12:30:15 EST 1994


In article <Pine.SOL.3.91.941123113627.19327A-100000 at dale>, Yee-Yung Charng
<szcharng at peseta.ucdavis.edu> wrote:

> Hi, I wonder if anybody can give me a clue to make ssDNA of pET-based
> plasmid with good yield.  We have tried Novablue and XL1-blue as host 
> cells and USC-M13 as a helper phage without success. I was told to use 
> Promega's R408 helper phage instead.  Any input ?  Thanks in advance.
> 
> Yee-yung Charng
> yccharng at ucdavis.edu  

No ideas, but why would you want to?  For sequencing, we've had v. good
experience with ds-DNA including for pET.  For mutagenesis, we use a method
called recombinant PCR (I can find the ref. if you need it) which has given
very good results w/ bluescript and pUC; I don't know that anyone in our
lab has used it on pET.

Don Jackson



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