I used gateway cloning to clone my insert into the donor vector. After having colonies, I did a mini prep and ran it on the gel. On the gel I could see a clear band on the right length for my cloned construct but also a band at the length of uncloned vector control. Anyone know why there is an uncloned vector band and a cloned vector band in ONE bacterial colony?
There is a problem in one of the cloning stages that may be in electroporation, bacterial cultivation, or the capture of a single colony during the mini-prep that led to confusion between the plasmid-carrier and non-plasmid-carrier colonies.
Muhammad Haddad since I am doing gateway cloning and the cloning is based on recombination, do you think I can proceed with the sample, although it has full vector and vector with insert in there? In theory, it should anyway online recombine with the one w insert
This could reflect a mistake when picking the colonies if the plate is crowded, but most likely the reason is that a bacteria was transformed with two plasmids, one with the insert and one without it. If you have other recombinant clones, use them. If not you can transform bacteria with the mixed DNA and get new colonies trying to segregate both plasmids and use the one containing the insert.
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