deletion by PCR

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Mon Jun 15 10:57:04 EST 1998


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


Hi Andrew,
I guess it´s easier to do the deletion by a PCR "around the plasmid", rather
than trying to anneal  those two fragments.
That´s how it worked for me:

1. design primers, pointing with the 3´ end away from the region to be
deleted. 24mers are usually sufficient. If possible, design a restriction site
into both primers that does not cut in your vector and insert. If this is not
possible (e.g. because of reading frame considerations on fragments encoding a
protein), you´ll have to phosphorylate the primers before use (should not be
necessary for you)
2. use these primers in PCR reaction (approx. 20cycles, 50ng template DNA)
with a proofreading polymerase (or Taq if you dont care about the errors). You
might need to optimize the PCR by adding DMSO to 5% or formamide to 2%. Other
optimization might also work... You should see a band at the right size.
3. digest with the enzyme you had chosen in step 1 (don´t if using
phosphorylated primers with no site, of course)
4. ligate briefly (1h at rt in 40ul)
5. digest for 1h with DpnI to remove template
6. transform
7. analyse clones by minipreps. Yield usually >80%

Hope this helps,
Frank


> --
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> Dr Andrew P. Schrader
> Postdoctoral Fellow
> (Respiratory Research Group)
> Department of Pharmacy (A15)
> The University of Sydney NSW 2006
> 
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> 
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~



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