dam methylation? - digestion problem (Nov/27/2008 )
Hi, I need help with my cloning. I would like to clone a gene to pEGFP-C3 vector (clontech) using HindIII and XbaI restriction sites. I had no trouble to clone it to the pJET vector (fermentas) and to cut it from it. But I´m not able to transfer the insert to the double digested pEGFP-C3. As a result of my ligation and transformation I have lots of colonies but all of them contain pure pEGFP plasmid. Today I have checked the sequence of the vector using pDRAW and I realased that the XbaI is in the pEGFP vector dam methylated! But it wasn´t marked in the vector information sheet. I thought that just the sequence GATC is dam methylated and the pEGFP restriction site looks like TCTAGA TA. What will you suggest as control to prove the methylation really occurs at this site? Is it likely that there is a point mutation in the vector that changed the sequence TCTAGA TA to TCTAGA TC?
Thank you very much
Actually, I wouldn't think that it is very likely to be a matter of point mutation, although something like that is not impossible.
I would suggest you do two single digestions of your pEGFP-C3 -one with HindIII and one with XbaI- to see if both enzymes are able to linearise your plasmid. If they do then it means that there's definitely no dam methylation-related problem. If you find out that you really have a dam-methylated XbaI site, then you should probably use dam- E. coli cells.
the XbaI site in pEGFP-C2 is methylated. You should always check both strands for the GATC methylation site. You're correct when you state that the TCTAGA TA site will not be methylated, but the reverse strand does include GATC (as the XbaI site on the forward strand) is preceded by GA, so this results in a GATC sequence in the reverse strand, which is methylated.
Like f2dU says, you will have to use dam- E Coli if you want to use XbaI
Thank you very much,my mistake. Usually I like singing "always look on the bright side of life" from Monty Pythons in the lab. Now I should change it "always look to the wrong side of dna".