Molecular Cloning - Subcloning (Jun/20/2008 )

Hai,

I am trying to do subcloning in pBR322 plasmid, i have total of 6 fragments to insert, i did the first four very easily but am struggling for over a month to insert the fifth fragment. My 5th fragment 93.4kb) was cloned in TOPO vector and i digested it with DraIII and SacII enzymes with much difficulty but with success, so also i digested the plasmid pBR322 with the same enzymes and ligated the 5th fragment, got some colonies and screened them using Miniprep isolation method, my plasmid DNA concentration was very low in the miniprep. Also i didnt find any insert...can anyone explain why? is it because the size of the plasmid with four fragments is already too much for it and hence the addition of the fifth one is causing a problem...however there was no fifth fragment positive plasmids so far...then why is the miniprep concentration very low..is it to do with the bacterial competent cells...but i have been using the same stock of cells for inserting the other fragments...can anyone please help me...Thanks in advance

I don't know if the size you listed is a typo, but if it is not, then you have a problem. 93 Kb is a very large fragment, and probably cannot be cloned into pBR322. You would have to use a BAC or cosmid cloning vector. Additionally, most of the miniprep and transformation techniques used for smaller plasmids will not work, or will work very poorly, for such large fragments. How large is the final construct you hope to make?

Not sure why that miniprep is so low compared to the others. pBR322 is a low copy vector though, so you would expect lower amounts of plasmids sometimes. Things you can do with low copy vectors are use less ampicillin in your cultures or try a method called chloramphenicol amplification. If your insert is 94 kb then this vector really is unsuitable.