I am working on a virus that we have in a bluescript vector. The end goal is to rtPCR patient data but to test the primers I'd like to use our viral genome without blue as some of the primers will span the blue vector as well making the product 3kb longer.
So I tried to cut it, run on a gel, extract the viral band, then ligate that. Then I run that against the cut viral band and an uncut plasmid that is approximately the same size as the virus, assuming the major band on the uncut plasmid to match the size of a single viral genome religated and supercoiled. Then extract just that band. Doing PCR though anything that spans the cut site isn't amplifying. The virus was put into the vector with the same cut site on both sides (BamH1)
Either way I can use the original in bluescript, but I'd like to have matching sizes between the test PCR and the background transcript in the patient data as a supplemental picture. My question is after doing T4 ligase, will denaturing the DNA break the strands at the BH cut site? Is there an easy way to ensure it doesn't break apart? We can't transform cells since we don't have a selection marker after cutting out bluescript.
I haven't had trouble with a vector falling apart at a restriction site after ligation; unless you're missing something in your buffers or incubation I don't know why that would be happening. you say you are not getting amplification from any primers that span the cut site- how much viral sequence beyond the BH cut site does your primer contain? how are you confirming the viral insert is successful? do you have viral-only primers that you can successfully run on the ligated plasmid?
I have 27 pairs of primers (some overlap) in both directions. Anything that spans the BH site doesn't amplify, anything that doesn't span it amplifies fine. When I use the original stock still in bluescript, all the primers work. So the primer locations don't seem to be an issue, although they aren't great primers as we don't have much of a choice in location and one of the major genes is basically a bunch of very long T strands.
Just as an update if anyone searches this question in the future. Something strange happened in general with our T4 ligation, the normal PCR bands I got we tried to ligate into pcgt7 vector didn't work either, and a researcher with a lot of experience in the field also stopped getting any of his T4 ligation to work as well. New kit, same problem.
Switching to infusion cloning worked, then got kicked from the lab due to a PI meltdown so not working on this problem anymore.
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