XhoI problems - (Mar/23/2006 )
I have a 500bp PCR product (Pfu polymerase) which has a XhoI restriction site on both ends. I want to ligate this insert into a pGEM-vector. Sounds easy but somehow its not working.
I took care that the vector is dephosporylated, I purified every fragment either by gelextraction or by column, I tried different vector-insert-ratios. I even sacrifieced two bars of the finest swiss chocolate to the lab-gods ! But somehow I never get any positive clones. After checking on the internet I have the feeling that choosing XhoI was a mistake from the beginning. Do you guys also have had bad experiences with that enzyme? If yes, how did you solve it?
have you done a sequence with your PCR product to be sure that it is what it's supposed to be?
other than that...there may be a bad enzyme or something; have you done all the proper controls to check the system?
the only other thing, yeah...XhoI sucks for many people in cloning applications. I don't know why, but even if it's an urban myth, it's quite persistent and the only real way to get past it is to design primers with a different RE
if it's possible, try to avoid gel purification for vector. For insert, do a column purif only.
Or 2nd way : use pure agarose.
I've made several clones with xho1 (in combination with Kpn1, vector pGL3). I purified both digested vector and pcr product (purified pcr, digested it and purified again, you need quite a bit of pcr product!)on a LMP gel recovered using agarACE. I digested over-night on both accounts.
I can't remember off hand but does xho1 require extra bases beyond the recognition site? I seem to remember that this was a problem and made my primers a few bases longer.
I've also noticed that this enzyme goes off quite quickly so you could try a new batch. Or change primers, they're cheap.