XhoI! really a bad site? - (Jul/30/2008 )

hi, I'd like to know if anyone has some expreiences in digestion of pGL3basic with XhoI site? In fact, unfortunately, my insert has only MluI and XhoI, so no other choice here. Iv tried to open pGL3 with XhoI and MluI. STEP1, digestion of pGL3 with XhoI for over 1 H, then iv runned the sample on gel agarose 1%, 3-4 bands which means the vector is not well digested. Anyone can give me some advices? By the way, the XhoI iv used is from Invitrogen, and according to the guide, iv used buffer 2.
Thanks a lot!

XhoI is a good enzyme, one of my favorites.
Things I would try in your situation:
- More enzyme (up to 2.5 ul for 50 ul reaction), more time for cutting (up to 24 hr).
- Control of DNA quality: cut pGL3 with MluI, see if it cuts the way you expect XhoI to cut.
- Control of enzyme quality: cut any other plasmid with XhoI.
Do all three, you will find the answer yourself.

The NEB website says that Xho I doesn't digest methylated and/or supercoiled DNA very efficiently, and recommend using up to 10x the required amount of enzyme.

Iv used 10U enzyme for 1µg plasmide.
I think that's enough, right?

I am curious about your "3-4 bands" thing. Do you run uncut pGL3 on the same gel?
If you do, you will see, which of those four bands is a cut plasmid.

Now, if you are saying that MluI does not cut either, than it could be:
- Either a DNA quality issue -The best way to solve it is to retransform the plasmid and make a fresh minprep.
- Or plasmid identity issue - To confirm that you indeed have pGL3 in your tube, cut it with, for example, BamHI - KpnI. You should get 2.8 and 2 kb fragments.