Stuborn band after digestion of plasmid DNA - (Jun/16/2006 )

Hi everyone,

I'm using alkaline lysis method for my plasmid extraction, the plasmid DNA extracted was clean with minimal contamination of RNAs and phenol chloroform according to the OD measurement using both Nanodrop, spectrophotometer, and gel electrophoresis. The problem is, I cannot get any complete digest even after long long hours of incubation time (overnight).

Gel electrophoresis shows the expected bands sharp and intense but always with a faint and distinct band on the top. I have also tried including more enzyme with recommended amount of buffer for further incubation but no improvement.....

The only thing I can suspect is during alkaline lysis and neutralization steps during the pDNA preparation. I'm using 5ml of Solution I for cell resuspension, 10ml of Solution II for cell lysis and denaturation of gDNA, and finally, 15ml of Solution III for precipitating SDS-gDNA complex.....

Can anyone help me out on this. Thank you so much.

Sincerely,
Haohao

just wondering if there is a chance that you have a second plasmid contaminating (that is not cut by your restriction enzyme of choice)????
perhaps you should try cutting with a couple of different enzymes and see if it changes?
is the cut band your expected size?

We often see these, and still do not understand them. These bands simply do not cut, but seem to have little influence in further work. Ignore them and go on.

It's unlikely to have contamination of other plasmid because I have tried out several enzymes on the plasmid DNAs (even double digest). The bands turned out to be the expected sizes but with the extra stuborn band on the top.

The stuborn band that cannot be cleaved do exist and shown when I run the uncut plasmid.

I've been thinking of this stuborn band might be some form of the plasmid that somehow its structure has been irreversibly damaged during alkaline lysis (where the double-stranded DNA starts to denature), and neutralization step (where the plasmid DNA renatured but not for chromosomal DNA).

Any ideas???

Yes this is due to irreversible denaturation of the plasmid DNA. There was a paper on this many years ago (I think Nucleic Acids Research or maybe Analytical Biochemistry) which showed why this occurred. The solution was to use either boiling-based miniprep or reduce the NaOH concentration down to 0.15M.

Daniel

Molecular biology protocols