ligation problems - cloning a PCR fragment - into a double-cut vector (May/08/2001 )

Hi All,Could any one please help me out with my problem? I have been trying to clone a 300 bp PCR amplified fragment intoa mammalian expression vector . The cloning sites are Bst98I and BamHI. the fragment is Quiagen column purified and the vector is gel purified. I have confirmed single digestions and can see the DNA on the gel before ligation which is carried out @ 16 deg overnight. On transformation into electrocompetent top10f' cells, I get more than hundred colonies on my 1:1 and 1:5 ligation ratio plates and NONE on my insert minus plate. My time constants for transformations are similar. We reuse cuvettes as they are so expensive, but I'm sure there is no contamination. All the clones I screened for were parentals. I cut them with the same enzymes as I had tried the cloning. Where do you think things might be going wrong? I am thinking of colony hyb, but isnt directional cloning supposed to be a lot less messier than this? SHould I try a different strain? Any hints? I'd be really grateful if you could help me out with this.

Thanks in advance

lakshmi

Dear lakshmi,

You dont mention anything about the PCR fragment except that it is 300 b.p. I assume that the restriction sites that you are cloning the fragment into are also in the primer sequences, when you amplified the original PCR fragment. I also assume that you cut the PCR fragment with these enzymes, Bst98I and BamHI first, then quiagen purified the DNA before cloning. How far into the primer sequences are these sites. If the BamHI is near the end of the primer sequence, at the 5' end, it probabley wont matter too much. But the other enzyme I dont know. If the Bst98I site in your primer sequence is too close to the 5' end, the enzyme may not be cutting all that efficiently. In which case, self ligate the PCR on its own, cut back with both enzymes (double digest?), purify DNA then reclone into your vector.

Of course, if the PCR fragment doesn't have these sites then your blunt end cloning the DNA into DNA vector with incompatible overhangs ! !

Regards

Dean

I routinely use the TA cloning kit from Invitrogen for cloning PCR products. After you amplify your band, reamplify it using only half the concentration of nucleotides. Then ligate 1-2 µl of the PCR reaction directly in to the vector. Hopefully some of the restriction enzyme sites will be compatible with the vector you wish to use.

As others suggested, first I would try putting the PCR fragement in any PCR product cloning vector, such as TA, PCRSCRIPT etc and then shuffling the insert into mammalian vector. Alternatively, can't you use a mammalian vector, thats a shuttle vector?. that way you could confirm your cloneeasily in the bacterial system, before getting into transfection, which is more cost effective. Also, insteadof using restriction digestion or colony hybridisation to screening, you might want to screen using PCR, with your specific primers. You need a lot of recombinant digested, in order to see your insertshowing up on the gel. i.e, if your vector is 5 kb, you need ~200 times more insert to show up on the gel. Otherwiseyou will be seeing only the vector on the gel. I do not suspect ligation at this stage, as your insert minus are all negative, and your double digests seems to be pefect.

Do you purify your PCR products by phenol-chloroform? It seems like your ligation reaction is contaminated with the exonuclease which may come from your PCR reaction.