Help needed for megaprimer mutagenesis using PCR

[Lori Bentsen]

I have been using the megaprimer mutagenesis method to introduce
EcorI sites in the pR' template in E. coli.  This method uses two Pcr
reactions to inroduce a mutation.  In the first PCR reaction, a
universal primer is used (T7) and the mutagenic primer is used to
amplify the DNA.  The second PCR uses the product form the first PCR
reaction (which should contain  the mutation) and a second universal
primer (T3) which should amplify the rest of the DNA.  We have been
getting strange results from the first PCR reaction which is usually the
most simple.  At first we were getting PCR products larger than the
template (from T7 to T3 it is 545 bp).  This does not make any sense or
seem possible.  We did identical reactions but changed the annealing
temperature from 50 to 55 degrees C and got products with multiple bands
one still being larger than the template used and one that is the same
size as the template (545).  The expected sizes should be around 300
bp.  The reaction  conditions that are being used have been done before
in the same manner and have been successful.  I would welcome any
suggestions.


                        Lori Bentsen
                        lbentson at uoknor.edu