Low sized bands in plasmid preperations - its not RNA! (May/13/2009 )

What are these low molecular weight bands (arrowed-above and below bromo phenol blue) in the recombinant plasmids isolated from E coli Top10 strain using Sigma plasmid isolation kit? We have tried RNase treatment which didn't work, so its definitely not RNA!

hey Ram - while your typical plasmid prep gives the characteristic 2 bands that you are seeing, there are actually about 7-8 different forms of DNA that are evident in an uncut plasmid prep.

first, the very lowest is possibly supercoiled

second, the smudgy band (upper arrow) sure looks like RNA to me. RNAse treatment is not a be-all, end-all.

I would do a restriction digestion and make sure you ONLY see the desired bands. this will rule out contaminating DNA. if it looks clean after digestion, you're good to go. of course, you can always sequence...

aimikins on May 13 2009, 07:31 AM said:

Thanks aimikins for the info...
Would the supercoiled plasmid go so below?
one more thing..these are the plasmids with the typical concentration of 100ng/ul. We prepared 10ng/ul solutions for some purpose and stored those at -20C for a month or so...Strikingly the upper bands and the upper arrowed thing disappred from these preparations and only the lowest one remained! Is it because of plasmid degradation?
Sequencing of these plasmids didn't work very well, thats why we went for checking those on gel...
Thanks
Ram

ram on May 14 2009, 07:25 AM said:

Hey, I really have strong doubts if supercoiled DNA will run so low...deagradation also appears unlikely, in that case i would expect a smear, not a sharp band. but as suggested by aimikins, why dont u do a RE digestion?

plasmid suffers nicking. fairly common . one nick cause it to lose its supercoiled conformation. happens if stored too long.

hanming86 on May 15 2009, 03:31 AM said:

we have preserved many plasmids in our lab in -20C/-80C without such problems. and really, till now i was under the impression that nicking in plasmid DNA is not all that common (unless of course, badly treated )! anyhow, do u think nicked DNA would show such a pattern? i'll b surprised....

We tried the digestion of plasmid with single cutting enzyme which resulted in a single band corresponding to the linear plasmid and an ambiguous band of lower size was as it was!

i preserved mine in 4C . sometimes just room temp. there's no reason to have supercoiled plasmid anyway in most cloning. save time when i wanna proceed with RE or transformation. no need to wait for it to thaw etc. u should be surprised coz we have seen it many times even in our typical GE class. maybe u got a great lab that just purify supercoiled plasmid. i dont know .. but in my experience it's always a mixture of supercoiled and nicked plasmid. very easy to distinguish when u run undigested and digested plasmid side by side.

fastest = supercoild 2nd linearize 3rd nicked.


regarding ram situation ,

"strikingly the upper bands and the upper arrowed thing disappred from these preparations and only the lowest one remained! Is it because of plasmid degradation? "

it didn't disappear ram, because u dilute the heck out of it , it becomes barely visible on gel.

hey hanming...
Although we diluted the plasmid to 10ng/ul, we used large volume-10ul of it corresponding to 100ng plasmid for checking on gel. So I don't think it is just because of lowered intensity due to dilution. If this only would be the reason then the lower band also should get fade!

This happened to me before. I used another batch of competent cell and solved the problem.