deletion by PCR

[Michael Blaesse]

Hi Andrew

if I understood your question right you generate the two fragments by PCR 
and you like to combine them in a third PCR reaction. That is the 
same method which I used for site directed mutagenesis. In this 
case I would say the linker is to short. I used a 20 bp overlap 
between the two fragments. I purified the fragments by 
Phenol/Chloroform extraction and Ethanol precipitation to remove 
the primers. 

Michael Blaesse


> Andrew Schrader wrote:
> > 
> > I have designed an experiment to create a 70 bp truncation in a cDNA
> > species (eventually to be used in QC-PCR). Two fragments have been
> > generated (either side of the region to be deleted) with the 3'-region
> > of the first being the same as the 5'-region of the second - a 10 bp
> > match using a linker on the 5'-primer of the second fragment. After
> > denaturation, I can not get the two fragments to anneal in order to
> > generate a long fragment.
> > 
> > Is there a paper/protocol that outlines this method and is my linker too
> > short to allow annealing?
> > 
> > Thanks
> > 
> > Andrew
> > --
> > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> > Dr Andrew P. Schrader
> > Postdoctoral Fellow
> > (Respiratory Research Group)
> > Department of Pharmacy (A15)
> > The University of Sydney NSW 2006
> > 
> > Telephone:
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> > Email:                                andrews at pharm.usyd.edu.au
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> > 
> > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> 
>