problems encountered when working on a large plasmid
Posted 17 December 2010 - 10:15 PM
I am recently working with a large plasmid(20kb, large enough for me). i use DH5a as host strain.
When I use mini-prep kit to extract this plasmid, and run agarose gel, I can observe at least three bands. one is the right 20kb supercoiled plasmid I think, the other two bands are larger than the supercoiled plasmid. So I cannot use this plasmid in downstream experiments.
I thought it is genomic DNA, however, NotI digest or EcorI digest can give right band patterns.
can you give me any suggestion?
thanks very much!
Posted 18 December 2010 - 06:48 AM
Posted 20 December 2010 - 01:21 AM
actually the largest band do not seems like relaxed or nicked band because it is so large that it can just migrate into the gel(0.6%). i am wondering whether it is multimeric plasmid.
ps, i was working with several plasmid around 16-18kb, this is the first time I encounter this problem. The method I use for extracting plasmid is the same.