DNA cloning problem - (Sep/29/2008 )

Hi,

I'm trying to clone a 4-kb PCR product into a 4-kb vector. I had successfully PCR the insert from genomic DNA and performed restriction cutting with 2 enzymes, kpnI and BglII. I did the restriction cutting with vector at the same time. I had done a control on the vector, i.e. (1) single cut without ligase and (2) double cut with ligase --> DNA transformation of the vector into DH5A competent cells. Both (1) and (2) had very few colonies (2 to 3) as background. Then, I used this "OK" cutted vector to ligase with my insert (16C, overnight) and then performed DNA transformation as usual. I got 20 to 30 colonies, I picked all of them and did PCR check clones. Non of them showed the 4kb target band. Then, I minipreped 10 of them and do a single cut on the plasmids, only one of them is 8kb, the rest were 4kb. Then, I sent this 8kb for sequencing. The result is this is an empty vector without insert!

I repeat the whole process for 3 times, the best I can get is the 8kb empty vector.

Anyone can tell me what's wrong with it? I cloned other PCR products (800bp, 1kb) at the same time and I could get the right clone (9 out of 10 colonies) in just one trial. I believe the restriction enzymes, ligase and competent cells are OK in my lab.

Also, what is the best method for me to check the right clone in my case - the insert and the vector are both 4kb in size?

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Did you dephosphorylate your vector? I'd highly recommend it in this case to prevent vectors from ligating together, because both your vector and insert are about the same size with the same sticky ends.



It's probably the fact that this ligation (4kb) is less efficient than the others, especially if you haven't dephosphorylated your vector.



Try to analyze the map of the final vector you want after cloning (i.e. 8kb) to see if there are cut sites in your vector and insert that you can use to generate a restriction map. RE sites that are in your insert but not your vector are especially helpful.

Ginger

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Dear Ginger,

Thanks for your suggestions. I will try them later.

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or you could order primer that will amplify the internal site of the PCR product ( 1000 bp or less?!) , then do colony lysis PCR subsequently.

i have done a ligation with insert n vector of nearly same size and got good result.

Did you leave enough extra bp for the RE to stick on to the PCR product?
tell us the primer seq that u designed if possible

thanks.

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