Cloning not working, all steps checked, clueless - (Feb/15/2013 )

Dear all,

I am trying to do a cloning of 15 truncation mutants into a impact system plasmid pTXB1.

The PCR was fine for all, yielding exactly the sizes I expected, the double digest worked fine, as single digest of each enzyme (SapI, NdeI) of the plasmid gave a nice band of linear plasmid, as compared to the undigested supercoiled mess.

The digest also worked on the inserts, which i saw on a 4-20% TBE gel.

The gel purification of plasmid and inserts worked ok, the concentrations were not too high, 10-40ug/ul, but the product had a nice 260/280 and 230/280 ratio, and was clearly visible on another high resolution gel.

For the ligation, i tried a double digested plasmid, as a negative control, a single digested (SapI) as a positive control, and took my best looking insert in a 1:1, 1:3, 1:6 and, opportunistically, with a 1:20 molar ratio. I used quick-ligase for 10' at room temperature. As stated by NEB, i didnt heat-inactivate it.

I also tried the enzyme prior to using it on a 1kb ladder. 10 minutes was enough to get more or less a single band.

As an additional positive control for the trafo, i took an uncleaved plasmid. I transformed 20ul of each reaction

RESULT: positive control worked, whole plate über-full, None of the others had colonies.

FACTS:

- The primers are known to work well ( i took the cleavage parts from primers which have previously worked on this plasmid)

PCR worked
Both cleavage steps worked
The ligase is active
the plates are fine
the T10 cells are fine


What i also did overnight is do the same ligations with a T4 DNA ligase, on 16°C, but seing as this is my third attempt, i am skeptical.

Question: why did the single cleaved, gel-excised plasmid not close? A single restriction is usually the reason we get false positive clones anyway...

Baffled

Thank you to anyone that can help, or try and help me

I'd suggest you have poor quality competent cells. Measure the competence by transforming 10 or 50 pg of a common plasmid such as pUC19. It should give 10**8 cfu/ug, or about 10000 transformants for a 10 pg DNA sample. Transforming successfully with 20 ng of DNA proves essentially nothing about the quality of your cells. You probably are using too much ligation product in your transformation. Less is more, and about 1-2 ul in a 50 ul cell volume is a good choice. Given you have cohesive ends, quick ligase typically causes more problems than it solves. I would use T4 ligase. You should be able to ligate for 5 minutes at RT. Another common problem is UV exposure during gel purification. Make sure you are using long wave UV or (better) blue light.