Posted 17 March 2005 - 01:52 AM
Has anyone ever tried to add XmaI sites at the ends of PCR primers for cloning? Something strange is happening to me:
I do a PCR adding XmaI sites on each side, run a gel of the reaction, and cut the intense band at the proper size (2.2kb)
After purification, I digest with XmaI and run the reaction on the gel again, and end up with two bands: one at 2.2 kb and one at around 4.5kb!
It makes no sense to me; I wouldn't give it too much thought normally, but incidently the cloning I do after that doesn't work, and it's the only cloning that gives me problems these days. So I am wondering if this strange phenomenum is not part of the problem.
Any experience or suggestions? Thanks!
Posted 17 March 2005 - 05:34 PM
Do you gel excise + purify your PCR product or do you just run some ul to verify your product?
If you don't gel excise your product I could imagine that you still have some of your PCR template in the mix. What was your template DNA? Does your template might have a XmaI site?
Have you tried to cut w/ SmaI? It's an isochizomere generating blunt ends (could make your subsequently cloning step more difficult).
Posted 18 March 2005 - 12:07 AM
Thanks for the answer; I do cut my PCR band from the gel, which makes the appearance of the upper band intriguing. I am quite sure it is not the template, since I add only 10 ng in my reaction, and in a negative control (without polymerase) I can't see it on the gel.
I tried with SmaI, and I had the same thing! Is it somehow possible that buffer 4 can sustain some kind of ligation, and that the upper band corresponds to two inserts tied together? The sizes would match, but I have never seen this before...
Posted 18 March 2005 - 08:30 AM
I can't imagine that you can do "ligation" with a enzyme buffer - you do need a ligase to do such things ;-).
Have you tried to change the enzyme buffer? Maybe the buffer is contaminated.
Posted 23 March 2005 - 12:04 PM