unable to clone into pET29b and pET32b - sticky end ligation failing (Oct/22/2007 )
Hi,
I have a PCR product with BamHI and SalI sites attached. I am cutting my vectors (pET29b and pET32b) as well as PCR product with BamHI and SalI. Then I am setting up the ligation at different molar ratios( typically 3:1 and 6:1 of insert to vector). I am getting barely 4-5 colonies and none of them have insert. Does anyone have a similar problem with pET vectors?
I am thinking of TA cloning the PCR product into a TA vector like pGEM-T and then cutting it out with BamHI and SalI and then ligating it to cut vector. Do you think this is a better approach?
Will appreciate any help. Thanks.
-lotus-
I think so. a;ternatively, you can use TOPO cloning kit.
For cloning into the pET vectors, did you check that the digestion is complete?
-Shirleyler-
QUOTE (lotus @ Oct 23 2007, 12:37 AM)
Hi,
I have a PCR product with BamHI and SalI sites attached. I am cutting my vectors (pET29b and pET32b) as well as PCR product with BamHI and SalI. Then I am setting up the ligation at different molar ratios( typically 3:1 and 6:1 of insert to vector). I am getting barely 4-5 colonies and none of them have insert. Does anyone have a similar problem with pET vectors?
I am thinking of TA cloning the PCR product into a TA vector like pGEM-T and then cutting it out with BamHI and SalI and then ligating it to cut vector. Do you think this is a better approach?
Will appreciate any help. Thanks.
I have a PCR product with BamHI and SalI sites attached. I am cutting my vectors (pET29b and pET32b) as well as PCR product with BamHI and SalI. Then I am setting up the ligation at different molar ratios( typically 3:1 and 6:1 of insert to vector). I am getting barely 4-5 colonies and none of them have insert. Does anyone have a similar problem with pET vectors?
I am thinking of TA cloning the PCR product into a TA vector like pGEM-T and then cutting it out with BamHI and SalI and then ligating it to cut vector. Do you think this is a better approach?
Will appreciate any help. Thanks.
I assume you've introduce BamHI and SalI restricition sites by adding them to the primers, can I ask how you've done this? To ensure digestion you need to have extra nt after the restriction site. So if for BamHI you add GGATCC to your forward primer, you should CCC or GGG before that, making your primer CCCGGATCCNNNNNN(N being your specific sequence).
I personally always clone in pGEMT first, is easier to confirme digestion.
Also, are you dephosphorylating your vector prior to ligation? I have clone different proteins in pET29c and have never had big problems.
hope this helps, Good luck!
-almost a doctor-