frameshift after ligation
Posted 03 November 2004 - 09:10 AM
Well, now the big main problem is, that we are not able, to cut out the insert of our positive clones, which have the insert for sure...! The blunt end restriction site MscI is no longer working. And so I am asking myself, is it possible that the enzyme MscI might cut a kind of sticky end with 1 bp overhang (however), so that the insert might be ligated with a single-strand break and the bacteria fill the missing base?!? Might the sequence TGGCCA be susceptible to mutations?
I would be glad for further information...
Posted 03 November 2004 - 04:51 PM
Edited by tfitzwater, 03 November 2004 - 04:51 PM.
Posted 04 November 2004 - 02:56 AM
But the point is that we have one base pair too much, so that the possibility of a exonuclease contaminant doesn't fit.
Posted 04 November 2004 - 08:57 AM
You don't have to use CIAP when you are using two different enzymes... alternatively, you could put the pcr product into TOPO and then cut out from there. Then you'll know you've got the right ends...
Have you tried to cut with EcoRI only and thus seen a shift in weight?