Another cloning hell....no transformants - (Nov/27/2012 )

Hej

I'm working on this project now since half a year. My overall goal is to make point mutants, but for that I had to construct a new plasmid.

This was supposed to be easy, because the insert was already ready made in a different plasmid. So I designed two primers with restriction sites (PstI and SalI) and amplified the insert via PCR and I got the product with the correct size (2700bp). The primers were designed that I have enough bps overhang for especially SalI cleavage (more than 5).
So I digested my insert sequentially (first with Fast Digest Sal I) and then with Fast Digest PstI.
The vector is in total 5700bp and SalI and PstI sequences are direct next to each other. Because Pst I can cleave with less bp overhang I digested sequentially as well with SalI first.
After ligation and transformation into electrocompetent E. coli. I never see any colonies (my controls work fine). I suspected my insert and checked if I can get dimers if I digest only with one of the enzymes. And both the Pst I and Sal) site work fine. Checking the ligation on the gel, I can see there is no ligation product.
So I think it is my vector due to the neighbour salI/pstI sites. My next try was to add a piece of DNA between them. Hence cutting only with PstI add a piece of DNA and then cut it out with salI and PstI (which are not next to each other anymore) and as a control I would see the cut out insert on the gel. So I used an insert with similar site and cut it with PstI and cut the vector with pstI. Ligated and transformed.
No colonies. I suspected my ligation, but on the gel I could detect a ligation product. So I repeated the transformation, but still nothing......

So It must be the transformation. I was thinking about raising the volume I add to my competent cells (Usually we add 2ul to 50ul electrocompetent cells). Or maybe purifying the ligation product with ethanol precipitation? I'm a bit clueless here and really would finally get this plasmid done......So thanks in advance!

I would definitely not choose SalI as an enzyme for this cloning. It has a bad reputation for cutting, although you say you have seen religation in the dimer test, so that is good. My first guess would be poor competence of your cells in transformation. Where do they come from?

My second suggestion would be to forget about SalI and to amplify the vector with some designed primers that would leave a friendly cut site. Choose good cheap enzymes that can be heat killed, like EcoRI or PstI, and make sure you leave sufficient 5' overhang on the primers. Amplify the insert with primers having the same cut sites. Purify the PCR product, mix in equimolar amounts, cut with both enzymes, heat kill, ligate, transform.