I have transformed NEB 5-alpha cells with a plasmid that I have pre-sequenced and confirmed it has the correct sequence. All I want to do is make more of this plasmid. However, when I transform NEB 5-alpha cells with the plasmid, the resulting transformants grow up on the Zeocin-containing media and contain plasmids that, when sequenced, show VERY low sequence similarity to the original plasmid. This has never happened to me before and I'm completely stumped as to what is going on! Can anyone help me out?
Before you panic I would suggest just trying again and screening a few colonies. It could just be that something was contaminated (such as with a different plasmid DNA) or you picked the one bad colony from your plate. Start with fresh DNA preferably from the stock you sequenced, transform and select a few colonies. I presume a quick restriction digest should be sufficient to demonstrate whether all are similar to your original plasmid or not before sending one or two off for sequencing.
In addition to Dr Benedik's suggestion, colony pcr from a bunch of the colonies would be a quick way to screen for potential correct plasmids and weed out anything that's contamination. You can probably devise a way to use your sequencing primers or any other primer pair unique to your plasmid to check the size of the amplicon if it seems right for your plasmid, it's pretty unlikely that the contaminating plasmid would amplify to the same size. That way you won't have to bother purifying the plasmid DNA from colonies until you have a reasonable assumption that they contain your plasmid.
I agree with Dr Kerman.
make a PCR on colonies and sequence the product to be sure that you have the good plasmid before amplification
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