Methods for in vitro mutagenesis
wind at biobase.dk
Thu Feb 6 10:01:07 EST 1997
Bernard Murray (bernard at elsie.nci.nih.gov) wrote:
: In article <32F8BA1C.167EB0E7 at bcm.tmc.edu>, kl690782 at bcm.tmc.edu says...
: >I am looking for a protocol for doing in vitro mutagenesis. I only need
: >to alter one base in the coding region of a gene but the problem I am
: >having is that all the methods Ihave been referred to are dependent on
: >unique restriction sites that flank the region you want to mutate. I am
: >having a hard time finding unique sites. Does anyone knopw of a
: >protocol where maybe I wouln't need unique sites?
: >Kellie Lugenbeel
: Stratagene's QuikChange kit doesn't need you to change vectors
: and doesn't require such unique sites - all you need is that
: your construct contains DpnI sites in it somewhere. I haven't
: tried the method yet but I intend to in the near future.
: [no affiliations with Stratagene]
: Bernard Murray, Ph.D.
: bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
I've had succes with Quickchange, its fast and easy (when things go right).
If you use it, introduce a RE site with your primers along with your
desired mutation. It will fascilitate the screening for positives, thus
the site does not have to be unique.
No affiliations etc...
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