restrict enezyme

Yuyuan Guo yuyuan at students.wisc.edu
Tue Mar 26 19:54:57 EST 2002


Dear Friends:

I have cloned one gene into TA vector. The ORF was with ClaI and SalI on
both ends. With specific primers, I can do PCR to amplify the gene from the
plasmid. This means the gene is in the plasmid. However, when I used ClaI
and SalI to cut the ORF, I could not get the band that is 1.1kb of the gene.

I tried other plasmid with other ORF with same vector and same restrict
enzyme ends, I can get the band.

As ClaI is methylation sensitive, I transform the plasmid into 110 meth(-)
E.coli.

Could it be that the primer was mis-formed? Like I order CCG, but they
formed CTG. So the restrict site is changed.

How do you think?

Thanks.

Peter





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