Making deletions with PCR

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Sat Feb 20 05:38:15 EST 1999


Hi Peter,
There is no upper limit for generating deletions by PCR. In fact, when
you use the method outlined below, the larger the deletion the better it
works. The key is to do PCR "around the plasmid". That´s how it works:

1. design two primers that flank the region you want to delete, but in
orientation that primers point away from the region. 
like this:


                             /
                  primer 1  /
               <-----------/
 ---------------------------I---to be deleted---I-------------------- plasmid
          
                                                 /------------>
                                                /   primer 2
                                               /
                                                    

In this drawing I have added an (optional) non-complementary region in
each of the primers to build a restriction site for re-circularization.
That´s not necessary for cloning, but very convenient. In case you
cannot tolerate additional bases at the site of deletion, just don´t add
such regions.
2. perform your PCR with a proofreading enzyme (Pfu is best in my
hands). Extension times are very long (2min per kb of your plasmid for
Pfu; i.e. 10min for a 5kb plasmid; I usually add 1 more minute "bonus"
(11min for 5kb)). Use 50 to 100ng of template, and 125-250ng of each primer.
(3. You might have to optimize PCR reactions to really get clones.)
4. digest your plasmid with the enzyme you have incorporated in the
primers (or don´t if you do it blunt-end)
5. clean up your PCR product, then ligate for several hours at RT
6. digest with DpnI (10units for 1h) to remove template DNA (this will
chop up you template because it is methylated DNA, but not your PCR
product because it is not methylated).
7. transform bacteria (electroporation or any other highly competent
bacteria will do)
8. pick clones for analysis (yield is variable, but 50-80% can routinely
be achieved)

hope this helps,
Frank




Peter Russell wrote:
> 
> How long of a deletion is it possible to make using PCR?  I have
> a need to remove about 140 bp and PCR would be a convenient method
> if it will work.
> 
> Peter Russell
> Reed College
> yeast at reed.edu



More information about the Methods mailing list