I am trying to clone a gene into a vector. After doing the ligation of insert and vector, successful transformation is confirmed by doing colony PCR of selected colonies. But when I isolated the plasmid and again did a PCR with the same primers as used in Colony PCR(for which I got Positive signal) I do not get any signal. What could be the reasons? What should I look into or modify?
When you do the pcr of the colony you also pick up small amounts of ligation reaction mix containing the pcr amplimer which then amplifies even though the ligation and transformation was not successful. Then you grow the cells but because the process failed there is no amplimer sequence inside the cells so no amplification. You are just detecting a wetting effect.
Try colony PCR with one vector-specific and one insert-specific primer. If you would get your desired product in gel, you can confidently say that your insert is present in the vector. The signal does not come from the ligation mixture of the plate.
Thank you for the reply, I will try to keep your advise in mind when I do my work.
If you do not have a vector-specific primer, then another thing you can do that, pick a small portion of the colony, prepare a patch plate, and from the patch plate you can do colony PCR with insert specific primers. Patch plate preparation is always good because it is free of ligation contaminants. Check your antibiotic also.
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