I have been trying to clone Sugarcane genes in the pENTR/D-TOPO cloning system. However, for almost of my obtained Kanamicin resistant clones I don't have success in the insert confirmation using M13 primers in colony PCR. What can I do to improve my protocol? In my protocol I used 0.5 uL of D-TOPO vector. Is that a common procedure for TOPO cloning protocols or Do we have a problem at this point? Anyone have a suggestion? Thanks !!!
Hi Helder,
with the limited information you provided is not easy to help you troubleshoot but there are two key points that could explain your poor results.
(1) did you use the apropiate primers for the directional cloning to work? Remember that you have to add the sequence CACC to one of your primers (forward normally), if not the directional cloning will not happen.
(2) did you use regular Taq polymerase or a proofreading one? For this approach to work you need blunt ends and a regular Taq won't produce those.
If you did design your primers correctly and you used Pfu or similar I would reconsider the ratio between your insert and the plasmid.
Hope this helps,
Ezequiel
Hi Ezequiel,
I'm very grateful for your tips. Answering your questions:
(1) Yes, I designed my primers adding the CACC sequence at the 5'-end of my Gene.
AND ...
(2) I used a high fidelity DNA polymerase (Phusion) to produce an blunt end amplicon.
I will quantify again my PCR samples to check the concentration of DNA. Maybe there is any mistake in my NanoDrop analyses.
Thanks,
Helder
According to my experience, sometimes colony pcr dosent work. Better isolate plasmid dna and follow restriction digestion to check on the agarose gel comparing the size of the bands. Its very reliable.
I am using clone-tech infusion system and its very effective.
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