transformation issues with ligation product - a double-digested (HindIII / HpaI) vector won't take up an insert (Sep/25/2008 )

Hi folks, I recently discovered this forum and therefore now have my first problem, which couldnt be solved by searching the archives yet.

I am trying to clone a pGEM-T vector containing a 3kb insert into DH5alpha. The insert contains a HindIII and a HpaI digestion site which shall be used to replace the contained gene with another gene from another pGEM-T vector which is flanked by the same digestion sites.

So i double-digest my 2 vectors, seperate the stuff on a gel and cut out the open vector and the desired insert. Then I ligate these two products and transform the ligated vector in DH5a, and here problems seem to start. I got a lot of transformants and screened some hundred until now, but all of them where negative concerning the new insert.

I never had problems when transforming DH5a with the pGEM-T Kit, since I had no problems getting my fragments into the pGEM-T vector. I already examined the digestion process which seems quite complete, loaded the digested and cleaned product on a gel again, to look for additional bands, I tried 1h and overnight digestion and ligation, I varied the vector / insert ratio and so on..

I know that there are some issues when using blunt end products, but I thought this might not occur in a dramatic way since the other enzyme is a sticky cutter. Currently I am creating new competent cells (cheers to the forum for the protocols) and hope to obtain my clone finally. But I am a little desperate since these problems do not seem to be impressed by any of my solution strategies.

Anyone got some more ideas to think about? Thanks alot..

Hi Picard

your problem can be solved if you ensure whether the vector in which you want clone the desired insert is properly digested with both HindIII and HpaI. Make sure to keep a vector alone (double digested with both HindIII and HpaI) as a negative ligation control. If you observe colonies with vector alone (double digested) ligation and subsequent transformation then vector is not completely digested. This may occur as you may be using a buffer if which one the enzyme is not haveing 100% activity. Since u are getting coloies and none positive then probability of vector being single digested seems to be more.

Well things would be different if you were sure if vector is properly digested with the enzyme and still not getting any colonies. then try using PEG 4000 (5%) in your ligation mix.

:)Zest

Treat the vector with a phosphatase to stop re-circularisation. Test the ligase by adding some to a DNA ladder and seeing what happens to it. You only need to ligate for ~30 minutes at RT, then run out on a gel (remember to also have a lane of untreated ladder!!); the ladder should shift its migration pattern up. You can also test your insert in the same way to make sure both sites have cut well. If you don't get a ladder with at least three rungs, one of your enzymes isn't working well.

Thanks alot already folks

one of my colleagues also suggested today to keep a negative control with the vector alone.. I'm gonna try that next week, but as far as I can determine, the vector got well digested with both enzymes. I will tell you as soon as I have colonies, wether the negative control created colonies or not.

The ligase testing with a DNA ladder is also a very good idea, thanks, a test of 4 different ligases went down the bin due to a bad bottle of LB..

Have a nice weekend everyone..

Back again, folks..

I have had little success this week. I didn't observe any colonies transformed with the digested vector alone in the ligation mix without an insert. And I also loaded a ligation on a gel and only observed the three vector bands way above the 1k-ladder as they used to run (the vector has about 6kb with insert).

As far as I can say, the vector is digested and ligated properly. Still, I got some colonies from a transformation, but still negative, and the control PCRs delivered some odd results even with the purified vector from the bacteria. I pretty much can't say what entered the bacteria.

Still, the colony number is pretty low (about 1-2 colonies per plate at 100µl / 1000µl transformation mix), and I only get them with my new competent cells, our old DH5alpha stock doesnt deliver colonies at all.

I now try a different strategy. Since I observed that transformation of pure vector yields in almost always very good results, I'm going to try to extract the vector from the ligation mix and transform it directly to the bacteria. My tryout today looked quite good, but I only did it quick and dirty (precipitate the DNA with ethanol, wash it twice and dissolve it in water), but the nanodrop showed an unrealalistic yield in DNA (10x as much as I put into the ligation), so I'm not sure wether to add a PCI extraction... I'd be glad about some comments about this plan..

and further ideas are welcome as well, the plan of a cleaned vector is rather a desperate idea than a real way..

oh, and by the way, I observed that in "Molecular cloning", the heat shock time is 90 seconds, I always used the 45-50 seconds delivered by the promega protocol about their pGEM-T vector system. could this also be a way to improve transformation quality? I somewhat feel like a master stud in his first semester asking that, but somehow I am currently grapping every straw..

Well folks, I managed to get the clone problem done, it was solved by a combination of fresh high competent cells and furthermore by extracting the ligated vector with phenol/chloroform from the ligation mix.

I don't know why, but the crude cleanup (only precipitation with ethanol) didn't help, only the PCI-precipitation yielded after transformation in 5 colonies from 4 plates, where 3 where positive.

So I don't know wether you can suggest this protocol for any other difficult transformation, but in my case it worked..