plasmid can't be digested by enzyme - (Apr/24/2002 )
E.coli Top10 have dam methylation ?
i have a problem with my double digestion as well.i have cloned a1.4kb PCR product into 5.3kb pET VECTOR.after plasmid prep. i double digested my vector with Sal1 and Xho1 but the plasmid is not digested.i cloned the same fragment in TA vector and digested using the same enzymes but there i got the insert aswell as the insert.i dont know where the problem lies.i am again doing plasmid prep yo check if the problem lies there.
any suggestions on this is most welcome thanks.
Man, you are good!
My construct has two Xba I recognition sites, however, only one of those has the sequence 5'-TCTAGATC-3' in the complementary strand. I used One Shot Top 10 F' cells to isolate the plasmid. After RE digestion with Xba I, instead of the expected 2-fragment product, I was able to obtain a linearized plasmid.
This shows that Invitrogen's Top 10 cells (packaged along with the pCRII-TOPO kit) has dam methylation activity!
First off, for anyone who does molecular biology, the NEB catalogue, believe it or not, is a bible. It has tables and charts with answers to questions you never thought of asking but should have.
So, first thing I see is that XbaI and NheI have compatible cohesive ends, so they will ligate together but they cannot be cleaved back apart by either enzyme. I can't tell exactly what you are doing but that might explain your ligation but uncleavability in one of your constructs.
The only one of your enzymes blocked by Dam is XbaI, but ONLY if the restriction site is 5'- tctagaTC -3' in EITHER direction. The small letters denote the recognition site, the caps represent non-recognition site nucleotides directly adjacent which form a Dam methylation site.
Hope some of that helps.
YES it does have dam methylation activity!