Hello everyone,
I'm a PhD student producing recombinant proteins amplified from genomic DNA and I'm having issues with cloning. I'll explain briefly.
I'm amplifying DNA sequences from genomic DNA with custom oligos that include the region of interest and are flanked with restriction enzymes cleaving sites to allow the insertion in pET vectors. This is working great, sequences have been checked by Sanger sequencing and are correct.
When ligating vector and inserts, and then transforming is when the problem arises. I'm getting empty vector back from the transformation. We have changed everything in the lab, from reagents, to new enzymes. And the results have gone from no-colonies-plates to a few colonies of empty vector. These colonies were checked by colony PCR previously and a positive result was shown.
Please can someone recommend any controls I can use to identify where the problem can be?
I have checked the digestion process of the vector with the double digestion and single digestion, and is working well, but don't know how to check digestion of DNA inserts.
Thanks in advance
Fidel
There can be several problems. First is the ligation itself. Sometimes the ligation is the bottleneck of cloning process. Vortexing buffer and ligation enzyme is sometimes crucial and should be performed according the the protocol. Further, if the cells are competent or compatible with the vector is also important. Also, the concentration of antibiotics in plates should be carefully checked. One can also try to use IPTG/X-gal to get blue white screening.
People can only speculate what is wrong with your cloning process, but most probably you need to troubleshoot the ligation step.
Which cloning technique you are using for ligation? I mean restriction cloning or gibson assembly?
(you can try infusion technique, gibson assembly to have seamless insertion).
however, you can run the vector plasmid(after digestion) on the gel (usually i run 1ul) for checking the right concentration for ligation reaction. if the band size is same as your expectation and concentration is considerable (it definitely should not be smear and not very dull) you can use 3:1 ratio of vector to insert. (you can increase concentration of insert based on insert band). also do you do purification of digested products prior to ligation reaction? i mean with after digestion the restricted product should be suspended in chilled isopropanol, and centrifuged and washed with 75% EtOH. (both vector and insert). if you do so then do immediately ligation setup as flanking ends can be degraded may be due to some RNAse type activity. are you using T4 DNA ligase? if so do did you completely dissolved the ligation buffer at RT? if so DO NOT VORTEX the ligation mix after adding T4 enzyme, but a gentle pippitte can work to do mix evenly. follow the protocol and then do transformation. for transformation, if you are using self made competent cells then assure their reception of plasmid from your past experience. transformation have several key points, like heat shock timing and shaking in LB without antibiotic to activate the gene expression and cold shock after heat shock etc. you can find relevant literature for optimization of transformation. good luck.
Abhijeet Singh thank you so much for your response. I agree with you, I think it’s the ligation process, but what specifically? Because my group have been using the Roche Rapid Ligation Kit for years and they haven’t experienced any problems. Also, past year I was doing some similar experiments and they worked. What baffles me is that the colony PCR with the specific primers for the inserts give me beautiful bands in the desired size, and when I send them for sequencing it returns empty vector.
Have you considered treating the vector with a phosphatase (CIP) before ligation ? The abscence of terminal phosphate groups prevent the ligase from self-ligating the vector and generating empty vector colonies.
Colony PCR directly from the plate is not entirely reliable as there is a possiblity that you might include a bit of agar with the insert from the ligation mixture. Its better if you can isolate the plasmid and check the construct spcifically for the insert instead.
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