PstI cutting - (Aug/09/2004 )
I want to cut a PCR-Product at 3' with PstI. In the NEB-catalogue they say that cleaveage-% of a sequence like aaaaCTGCAG+14b is >90. What about a sequence like xx1kbxxCTGCAGttaa, with extension at 3'.
Short: Can i cut directly th PCR-Product or is it better to do a subcloning step?
Did somebody do that?
How could you subclone if you don't digest the PCR product of yours???
Usually give about 4-5 bases upstream and downstream of your restriction enzyme's site. That should be enough fo the vast majority of enzymes.
Hope this helps.
Thanks for your answer!
>subcloning via TA-Vektor (TOPO)
>I know about adding bases 5'/3',
I ask wheather somebody did a direct digest like that (eg. xxx1kbxxxCTGCAGttaa) with Pst1
(With Sal f.exp. I had a lot of trouble cutting (+ligating) when only 4 Bases were added downstrem, but no Problem cutting (+ligating) when i previously made a subcloning via TOPO-Vektor)
Of course you wouldn't have any problems cutting a piece of DNA off a clone...
I would recomend trying to digest the insert as is and see what happens, it'll only take an afternoon.