Hi,

I work with voltage-gated sodium channels, which are a pain to clone into high-copy number plasmids. Recently I have acquired the pBI-CMV1 vector, which is a low-copy number vector, and it has really helped with the cloning. However, when it comes to DNA purification, I have had lots of issues. Sometimes the DNA yields are extremely poor (I mean barely detectable by Nanodrop), despite longer growth periods and increased culture volumes/lysis buffers (Qiagen) compared to high-copy number plasmids. What is most worrying is that I often get a high level of "smeariness" when I run miniprep/midiprep pBI-CMV1 DNA on an agarose gel. It's not one distinct form of contamination; it's more of a background smear/smudge that stretches across all DNA sizes on the gel (and this can be seen even without adding restriction enzymes). It makes Nanodrop yields deceptively high and ruins the DNA for downstream applications like transfection. I'm not quite sure what's causing it, as I've never seen it with my other cloning/expression plasmids. I do not use endA+ bacterial strains for cloning. Has anyone worked with pBI-CMV1 before? Or has anybody encountered a similar problem with a different plasmid and been able to resolve/minimise it?

Thanks!