Kpn1 restriction enzyme - (Mar/06/2007 )
Hye everyone.. I wanted to use this Kpn1 enzyme for my cloning project.. but then my supervisor told me there would be some complications in using this specific enzyme but she couldnt remember exectly what is it... So she asked me to go and check it out.. So far I've found that this enzyme is not sensitive to any methylation, and it has only one isoschizomer which is Acc651.. So I'm not sure what exectly the problem is.. I need to use this enzyme since it has the only unique site at the desired location in my vector.. So, does anyone have any problem using Kpn1 for cloning before?
I have used this enzyme a few times with no problems!!, I don’t remember any special complication.
The only point I could think is that you can’t inactivate it by heating, but I do gel purification any way, so I don’t think it is a problem.
Any other opinion?
Kpn I can be a little fussy when it comes to its buffer. Depending on which company you use, it can sometimes be difficult to do a double digestion with Kpn I. But apart from that non-problem, and the above suggestion, Kpn I is fine. In fact, I quite like it because it creates 3` overhangs, which makes it incompatible with most of the other common sticky end cutting enzymes which produce 5` overhangs.
one of my lab mate also reported that KpnI is complicated
sometimes he got wrong band in gel from DNA digested by KpnI
I have been using KPN1 for cloning into various vectors and never had a problem so far. Its actually an enzyme of preference for me. I always use NEB enzymes.
I've also never had any problems with KpnI as a restriction enzyme, also never had problems with double digest. 3' overhang could be the problem, but if both vector and insert are cut with KpnI, I don't see any problems why you should switch to another enzyme.
Kpn1 is an efficient enzyme to digest your DNA of interest. However, when it comes to ligations, because KpnI cuts with to give a sticky 3' overhangs (instead of the most common sticky 5' overhang), this is not well tolerated by the ligase, which can result in inefficient ligations. There is an isoschizomer of KpnI available (ie. it recognises the same sequence but it cleaves in the opposite orientation) - Asp718 (Roche), which overcomes this problem. Personally however, I find Kpn1 to work fine anyway.
I also never have any problem with KpnI.
Among all the common REs, I would say that I don't like to use NotI. Unlike other REs, which are 6 nts, it recognises 8 nts. If you are not careful you may have a frameshift.
Have you checked the NEB website for FAQs about KpnI? It is pretty good if there are any common problems.
I've found that Kpn1 from NEB often loses its activity, and rather quickly.. never knew why!