help for site directed mutagenesis

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sun Feb 25 21:48:10 EST 2001


Hi Keren, 

most strains (like XL1 blue or 298) make methylated dna (they 
always do, when I started mutagenesis business, I didmn't know 
either). This is important because it gives you a means to 
discriminate template DNA (methylated because it is produced in E. 
coli) from DNA (mutated plasmids) generated by PCR (is not 
methylated unless you buy (quite expensive) methylated nucleotides).

DpnI is a 4base cutter cutting only methylated DNA. So it will cut 
the template in the PCR mutagenesis reaction into nice little chunks 
(the recognition sequence statistically appears every 4*4*4*4=256 
bases) that won't harm anymore but leaving the mutated DNA copies 
untached. This will save you laborius purification steps and makes 
one major advantage of this protocol versus the single strand 
mutagenesis using bacteriophages.
When you transform the PCR reaction directly into E. coli, then it 
only will propagate (in theory, in fact there always will be some 
template DNA left, so you have to screen some colonies) your desired 
mutants.

Wolfgang





> > Wolfgang,
> Thank you for your fast reply.
> Why should E coli made methylated plasmid? And why DpnI?
> Thank you for all,
> 

-----
Dr. Wolfgang Schechinger
Lab N233 (Box 78, c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
Taipei 115
Taiwan R.O.C.
Tel +886-2-2798-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
wolfsc at ibms.sinica.edu.tw
-----


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