Insertions/deletions at cloning restriction site - (Mar/17/2006 )

Hi,

How can I minimize short (3-4bp) insertions/deletions at ligation sites which I find in many of my screened clones (at Acc65I site)? Can CIP be the culprit here?

Thank you for any suggestions,

Anetta

I've had the problem of short deletions with EcoRV. I was using SAP rather than CIP, so I wouldn't think the dephosphorylation enzyme was the culprit, unless they all have that problem. I do know that EcoRV has been shown to cleave off nulceotides when cutting, hence generating deletions. This may be a problem with Acc651 as well. The recommendation I had was to switch to Eco321, an isoschizimer of EcoRV, which doesn't have this effect to the same extent. You could maybe try switching cutters to KpnI if that is an option.