I was using pgem t easy to ligate my gene (500bp respectively), blunt end
and gel extraction product. The case is : because of lower concentration of DNA template for pcr, so I re-pcr my desired gene, then I do gel extraction. I ligate 16-18 hours, and I find blue and white colonies. But when I check my insert gene using pcr, there is no band. I use M13 F and M13 R to check my insert gene in MCS. Can you explain to me how to increase the efficiency of blunt-end (there is A tail because I use gotaq) ligation? And can I assume that my gene not stick to MCS?
Hi Diah,
There are several reasons why these ligation may be failing.
One thing to try, is to set up a ligation with 1ul of the PCR reaction straight, rather than gel purifying it. At what temperature do you perform the ligations? I would recommend leaving the reaction at 15C for 16 hours.
Is the pGEM-T kit you are using past its expiry date? Do others in the lab use the same kit for cloning and do they get their clones? Which gel clean-up kit are you using and do you ensure that you remove all alcohol after the wash step by doing a second centrifugation?
You refer to "blunt-end" twice and you mention that the fragment should have A-tail because you used Go-Taq. Why do you mention blunt-ends ? If you amplify the product with Go-Taq, it should be A-tailed, and the standard pGEM-T vector is T-tailed so the two can just be ligated together. If you are at any point blunting the product (insert) or vector this may be the problem.
You say you use M13 primers to check for inserts, but "get no band". Do you have a positive control for this PCR to ensure that your PCR reaction should work if the template is correct? Ask someone in your lab if they have another pGEM clone which they've made previously to use as control template for your PCR reaction. If you can detect the insert they introduced in that plasmid, you know all is well with your PCR reactions and you should be able to detect your clones if they are correct. If the issue is with the PCR, why not just try doing a plasmid miniprep (alkaline lysis) of your potential clones and digesting them to see if the insert is there.
Blue white selection is not perfect, especially with small inserts and you may find that the blue colonies actually carry the insert. Have you tried checking a few of the blue colonies to see if they have the insert?
A final thought is whether or not the product you are trying to clone may be toxic to E. coli when expressed? Depending on the orientation of the insert (TA-cloning so it can go in, in either direction) the gene product may be expressed off the lacZ promoter and kill the host.
Regards
Lonnie
Sorry for unclearly explanation. I assume my gene lost A tail because of 2 month storaging in -20C. And my friends told that my insert may lost A tail because I use uv too long (maybe), or nuclease contaminated. So I confused to said that it is still have A tail or not.
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