Cloning into pUAST - Help needed (Mar/27/2005 )
For the past few months, my aim was to insert 4kb long gene currantly found in pBlues into pUAST, using 2 restriction enzymes: NotI and XbaI. In the begining my insert seem to logate back to the template, therefore I used all different strategies to purify my gene )eg. gel extraction). With this I obviously loose a lot of DNA, but not completely. Following the ligation and transformation: I , now, do not have any clones. I would appreciate if anyone who did similar work, or already has the experience working with pUAST, give me some feedback.
I would advise you to gel purify your insert once you cut it out of pBluescript.
If you are having problems with DNA yield, increase the amount of DNA in your digestion.
I usually use 5 uL of DNA from a miniprep, do a 50 uL RE digestion, then run the whole reaction on 2 lanes on a gel. I then cut out bands from both lanes.
I would use only one gel extraction column for this, you will have to make up tubes to balance once you do the centrifugation steps.
Also, make sure that you are cutting pUAST completely with BOTH enzymes. Since these two restriction sites are close to each other, there is no way to tell that on a gel.
Make sure that the REs are in their optimal buffers. Not I and Xba I don't have the same optimal buffers so you have to work around that.
My general advice to you would be: do not try to cut corners when doing these experiments. You have to do gel extraction, you may even have to do sequential digests.
well first of all, thnx for getting back to me. Pritty much, everything you said is what I am usually doing:
Sequential digestion with XbaI and NotI, initially starting with 20ul of miniprep DNA, of both my insert and my vector. Furthermore, I run them on gel, and do Gel extarction using Qiagen kit, release DNA in final volume of 30ul. Run another gel, just to make sure that I have enough of DNA for ligation, thus I load 2ul of DNA. Final concentration is not the greatest, i.e. bands are very faint, but you can still see DNA that should indicate that I have something to start ligation rxn. Ligation, I set up final volume of 10ul, 1ul of ligase, 1ul of ligase 10x buffer, and the rest is DNA: I try to get 3:1 ratio, so I usually use 3ul of pUAST and 5ul of insert. I leave them at 4 degree, over night. I obviously set up ligation control, that is usually ok, indicationg that ligation conditions are OK, but I never got any colonies for my cloning. It's very strange.......and I tried many different things, altering the concentration, changing the ration, even not to use gel extraction: in which case I get religation of my insert into pBlues, so I have no choice but to do gel extarction.......And I am pritty much desparate. I looked for an appropraite litterature, and I could't find anything relevent. Thus, if any of you have any idea....anything different that I just described...get back to me
pUAST is big on its own. I use 100 ng vector for my ligation.
I usually spin down my ~1mL transformation culture, resuspend in 500uL SOC or LB. Then plate 200 uL. I'm basically doing a 'high density plating' so to speak. Because in my experience, I really get few colonies with pUAST. Average colonies I get are around 5-8.
keep me posted.
I also had difficulty cloning genes into pUAST for about 6 months! I was cutting the pUAST vector with 2 restriction enzymes and then cut/purify a ~9 kb band from gel using the Qiagen kit. Thereafter I did the ligation and transformation, No colonies!!!
Finally, after 6 months hard working, I was told by a senior lab technician that the gel purification process can sometimes interfere with the ligation/transformation. Next, I skipped the gel purification step and the transformation eventually started to work!!
So here is my advice to you. <<<<<<DO NOT CUT THE BAND OUT OF THE GEL>>>>>
After digestion, simply purify your plasmid or insert through the column (I used the Qiagen kit) and then proceed with the ligation and transformation. You will get lots of colonies but not every one of them will have your insert (approximately 1 out of 20). If you want a higher yield of positive colonies, re-digest your insert/plasmid for the second time and hopefully you'll get more positive colonies this time (about 4 out of 20).
Try it this way for once... I promise you that you won't regret trying it.
With best wishes,