restriction digestion of plasmid - (Jul/24/2008 )
I did a restriction digestion of plasmid pcDNA3 with PvuII.. on running on a 0.8% gel i was supposed to get two bands... around 3kb and 1kb.... i got these bands but i also sort of a band just immediately out of the well!!!!! wat could that possibly be??????
Could be undigested plasmid - maybe several bands with supercoiled, nicked and incompletely digested plasmid?
Did you run the undigested plasmid as a control on the gel?
I think the same as above. I always run uncut plasmid with my digested products.
If your plasmid prep wasn't done carefully enough, DNA right near the well could be genomic DNA contamination.
don think it was genomic DNA contamination... it looked relatively clean with no streaking... but there was a bright band near the well!!!! Even i thought initially that it cud be the undigested products but that too would run down in the gel right? why was it near the well?
high molecular weight substance, that is why it is near the well. As swanny has mentioned, it could be genomic DNA. Streaking in the gel from genomic DNA contamination, need not neccessarily be seen if the DNA streaking is light and even.
Can you post a picture of the gel?
Was this a mimiprep? It's a fairly common thing to see bands at the wells (see, for example, the picture at the bottom of this page). Not sure what it is, perhaps polysaccharides?
I think that you need to post the picture of the gel, some times I have observed some bands close to the well, but I do not believe that is genomic DNA, may be some protein or polysaccaride... or both