13 replies to this topic

[ram]

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!

[aimikins]

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...

[ram]

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

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...

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

[Vini]

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

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

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...

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

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?

[hanming86]

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

[Vini]

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

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

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....

[ram]

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!

[hanming86]

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.

[ram]

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!

[WHR]

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

[hanming86]

10 ul of 10ng/ul is 100ng.it's not that high. and most of the MW is contributed  by heavy molecular weight molecule ( the one at the high end) while the low end is small thus getting less binding to the extend that u can't see it.

[ram]

I just wanted to say that disappearance of the upper bands (relaxed plasmid) and persistence of the lower band in the dilute sample can not be because of lowering of the intensity due to dilution. Isn't is quite logical?  

[ram]

Problem located!
That extra band was coming from the lab-made loading dye; donno how We just loaded only the loading dye in agarose gel n the same band was there!  Can it be because of some microbial growth in the dye containing just glycerol, bromophenol blue and water?

[Vini]

ram, on Jul 19 2009, 07:10 PM, said:

Problem located!
That extra band was coming from the lab-made loading dye; donno how We just loaded only the loading dye in agarose gel n the same band was there!  Can it be because of some microbial growth in the dye containing just glycerol, bromophenol blue and water?

maybe its a DNA contam. That is, someone has used the sample loading tip in the loading dye and introduced the sample into the dye!