Detecting a one base deletion with SSCP or a PAGE?
wgschech at med.uni-tuebingen.de
Sun Aug 3 16:11:28 EST 1997
Hi, experts, hallo Bini!
In order to distinguish two plasmids that only differ in a one base
insert, I thought of using either a sequencing gel or performing a
single strand conformation polymorphism analysis.
The Details: I got a plasmid where I inserted one base using a PCR
point mutation protocol followed by digestion of the template with
DpnI and ligation/transformation of the remining mutated plasmid
Since I mutated five somewhat different
plasmids I'm looking for a simple and fast detection method, i.e. I
don't want to transfect all five plasmids (of each there might be 10
different minipreps to be sure to get a positive clone) to 293 cells
to see in a western blot if I get the right proteins.
It is possible to cut out a segment of about 400-800 bases where this
inserted base is located.
Thus, I thought of running the digests on a sequencing gel or on an
acrylamid gel after separating the strands. Might I see the one base
difference for sure? What method should I prefer?
A negative and a positive control is available (the unmutated
Any comments are
Thanks and Cheers,
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
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