So I have subcloned an 80bp insert into ~3kb vector that is flanked by EcoRV sites. The insert was made by annealing two phosphorylated primers into a blunted vector. I received my sequencing results to verify the insert is correct and now am trying to release this insert from the plasmid to clone into another plasmid.
Since the insert is so small, I digested 10 ug in hopes that the 80bp piece would be strong enough on the gel to remove it--in a 50 uL reaction with 2uL (40 U) of EcoRV for 3 hours and ran it on gel against undigested control. The undigested runs mostly as a supercoil; the digested plasmid has a clear and as expected very strong band around 3kb but nothing below that.
I have previously attempted to PCR the insert from a plasmid but this was somewhat unsuccessful as my negative control without DNA template, i.e. primer dimers only, runs nearly the same region as what may be PCR product but may also just be primer dimers.