I am trying to do the allelic separation of a ~8 kb region using cloning strategy with pKS plasmid. However, when I analyze the result of the colony PCR sequencing It seems that both alleles were not split into two different bacterial cells. Is it possible that E. coli has taken up two plasmids of that size (in several clones)?
NOTE: I have already seen plasmids purified on gel (there is only one insert) and the colonies were isolated on the plate.
A likely scenario if you didn't re-streak the colonies from the recovery plate, is that your colony PCR is amplifying DNA from both the colony and residual plasmid DNA that didn't get taken up during transformation and is still hanging out on the surface of your plate. I've definitely had this happen a few times, even when I was very careful to only touch the surface of the colony. Colony PCR is useful but it's also a pretty fickle and error prone technique.
If you think you have some positive clones I would either re-streak them and do your colony PCR on the isolated colonies, or just miniprep the plasmids and send the minipreps for sequencing instead of the PCR product.
A likely scenario if you didn't re-streak the colonies from the recovery plate, is that your colony PCR is amplifying DNA from both the colony and residual plasmid DNA that didn't get taken up during transformation and is still hanging out on the surface of your plate. I've definitely had this happen a few times, even when I was very careful to only touch the surface of the colony. Colony PCR is useful but it's also a pretty fickle and error prone technique.
If you think you have some positive clones I would either re-streak them and do your colony PCR on the isolated colonies, or just miniprep the plasmids and send the minipreps for sequencing instead of the PCR product.
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