Hi, I try to clone a V5-TurboID (1Kb) into a roughly 6kb big backbone (pEGFPC2). The restriction enzymes used are NheI and XhoI. Both fragments are gel purified and ligated with T4 Ligase (1h or 16°C overnight), then transformed to Dh5alpha. I can not get any colonies. Ligation ratios varied from 1:3 up to 1:10. Does anyone have any ideas how to solve the problem?
When you get no colonies at all this suggests some other problem. It might be that you did not actually recover vector from the gel, or that it was too heavily UV irradiated. It might a problem with the ligation reaction (buffer or enzyme) or it might be that your competent cells are not good enough. Run some appropriate controls and see if you can figure out where the problem lies.
Hello,
Several things can cause this problem.
1) Are you sure that your host cells were alive before the transformation?
2) Are your host cells competent cells? do you have to test them first with a vector?
3) Did the transformation process (electroporation or thermal shock) work?
4) Did the ligation work? Maybe there is a Ligase problem.
So I advise you to test the first 2 points, if your host cells are good, look at the 3 points.
Good luck,
Are you using the right antibiotic on the plates? In my experience when there is something not right and you didn't get the plasmid from Addgene directly, there is a mutation that is giving some issue. Sequencing plasmids is pretty cheap now. If you didn't get the vector from addgene, make glycerol stocks and sequence it. No colones at all, maybe try Top10 cells. I like the Roche Rapid ligation kit, if they still make it. Good luck!
Agree with all comments (Sofiane, Matthew, Michael) about the controls for vector antibiotic, competent cells/trafo efficiency (those can all be tested with transforming a low amount of your cloning vector), Ligase / Ligase buffer (ATP, DTT), keeping buffer as alicuots, sometimes adding atp helps.) Another thing: put everything on gel after purification to see if it is really ok and the concentration is right. Sometimes the reads from Nanodrop/etc are not very accurate for purified fragments especially if the concentration is low. Are you using the enzymes together in double digest? Can u see that both sites are cut? could check with individual enzymes to be sure. Good luck.
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