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keep getting incomplete digest for plasmid - (Mar/19/2006 )

Hi everyone,

I'm facing problems with my digestion on my pbr322 plasmid and keep getting incomplete digestion results. I extracted the plasmid by myself using phenol chloroform extraction and I doubt there is any phenol contamination in my plasmid as the ratio for the DNA measurement is good.
There is another problem besides getting incomplete digest, which is....

The size of the plasmid is 4361bp; however, I got a nice 7000bp band plus the bands of which the sizes I expected to have on gel electrophoresis. It doesn't make sense that the 7000bp band exist. If there was genomic DNA contamination during extraction of the plasmid, I would expect to get more bands besides just 7000bp for the extra right?

Please help me by telling me any possibilities that cause these problems...

Thank you very much in advance.




supercoiled DNA

nicked circles

contamination? (other plasmid)



well, i think that if it was genomic it would b like your say.
for checking of different forms of plasmid, put the loading gel in your sample (digested one) and heat 10' 56°. Briefly spin and load it immediately. Normally, the mutliple bands shoud be reduced to one (circularized vector) and your digested plasmid.
The other possibility is to gel purify you digested vector, ligation electroporation and miniprep on fexw clones to determine which one should be amplified for renewing the stock


Thanks a lot for the replies, really help out alot.
I will try the heating method to see if there is any improvement and get back to you guys.

Thank you very much again.