Detecting a one base deletion with SSCP or a PAGE?

Wolfgang Schechinger 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 
copies.
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 
plasmid).

Any comments are 
appreciated!

Thanks and Cheers, 
Wolfgang

-----
                                     
Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de

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http://www.medizin.uni-tuebingen.de/~wgschech/research.htm




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