Theresa,
For point mutations, we currently use MEGAWHOP, which is a
megaprimer-based whole plasmid PCR method. It has the advantage of using
single mutant primers of modest length (21-24 nt) in combination with
existing flanking primers for the target gene (either the 5' or 3'
flanking primers). We used to do a two-step megaprimer PCR to copy out
the whole mutant gene but don't bother anymore and just do a one-step
mutant megaprimer before doing the MEGAWHOP. The final product requires
DpnI digestion in-situ prior to transformation of E. coli. Our protocol
can be found at:
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocols&saved_msg=y#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCR
It's not elegant but it works. We'll have to try PIPE sometime. Looks
like it saves some time over MEGAWHOP at the expense of designing two
primers for each variant gene. I guess it depends if time is more
important than money or money is more important than time.
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 4/17/2012 5:53 PM, Theresa Hsu wrote:
Dear all
I would like to get some opinions on site-directed mutagenesis. What are the
current methods available? I know the Quick Change, are there others that work
better?
Thank you.
