Orientation of insert - (Sep/13/2008 )
I've cloned a PCR product into pCRII vector and been trying to use M13F, M13R and the PCR forward primer to determine the insert orientation. What perplexed me is that I actually got positive amplicons in both orientations from 1 clone. Apart from contamination, which has been minimized, is there any possible explanation for this? I would appreciate it very much if anyone who has had similar experience could tell me about her/his solution.
1- the colony is mixed. Both versions of the plasmids are present in your sample. The colony selected may actually be composed of cells from two colonies that were close together.
2- the primers are not specific. Due to none specific binding, side products are generated, which by chance appear similar in size to your desired product.
3- contamination, either at the template, or at the primers, or at the PCR mix.
I assume you have other clones which contain the desired plasmid?.
Discard this colony which gave this conflicting result.
does the sequence read back into the vector or is it just part of the insert? if it's a blunt end ligation, maybe the insert was inserted twice, once in both directions
Definitely a possibility, especially if the ratios of insert and vector had too much insert. Actually the story will still hold for sticky ends, as there is only the one cut site.
Have you done a digestion test? Cut the plasmid with REs either side of the cloning site, and see what size fragment(s) you get.