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
thanks for reply.
This is perfectly normal and you have a good DNA prep. Uncut DNA will always show multiple distinct bands. Relaxed, nicked and supercoiled are the most common with the supercoiled running the fastest. You should search the internet for "uncut plasmid DNA on agarose gel" for more information. If your digested DNA shows you the correct band pattern and there are no other bands, you can rest assure that your DNA is perfectly fine and can be used in downstream applications. I hope you saved your preps!
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.