I am so seriously screwed.
I'm still trying to clone my 300bp product into pBSKS+. Previously, I had used unphosphorylated vector , and had a lot of background, but with blue-white selection, I had a good shot of picking the colonies with the insert. Anyway....use CIAP, and the insert wasn't ligating. Went back to using unphosphorylated vector, 3:1 molar ration...yadayadayada....same as before....Now what has previously worked, doesn't. Getting really weird results as well, with the cut miniprep in some colonies looking as though they're not cut, and staying at ~10 000bp mark. pBSKS+ is only 2.9Kb. And my insert, 300bp.
I figured, maybe the enzymes aren't recognising the site... I was using BamHI and EcoRI, went outside these two with XbaI and HindIII. And there was insert in some of the clones. I expect bands at ~300bp....BUT i'm getting bands at ~800bp, and 2000bp. Where the hell are they coming from?
I haven't used another vector, because i can't get my paws on one, yet...
So, went back and recut some vector with BamHI and EcoRI (i'm not purifying this.... could this be the problem?) Went back and PCRed my insert with Pfu, purified it, cut with BamHI and EcoRI....Ligate, 3:1 molar ratio, room temp overnight.... Ethanol precipitate....electro transform....plate.... blue white selection.... pick positive colonies, miniprep.....double digest again, with BamHI and Eco RI....and also take another sample and double digest with XbaI and HindIII....run the gel.... yeah, some work...but really inefficient, and an ~800bp product, ~20000, product, as well as my ~300 insert, and the ~3000 bp vector in the sample I cut with XbaI and HindIII.
Can someone please tell me where i'm going wrong?
Other people in my lab don't purify their vector, and they had no problems.
I don't get it. I need help.... please.
Oh, does TA cloning work well with Pfu instead of Taq?
Edited by vetticus3, 11 October 2004 - 04:49 PM.