Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Low sized bands in plasmid preperations


  • Please log in to reply
13 replies to this topic

#1 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 13 May 2009 - 04:28 AM

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!
Posted Image
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#2 aimikins

aimikins

    Sunflower

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 117 posts
2
Neutral

Posted 13 May 2009 - 06:31 AM

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...
"it is a miracle that curiosity survives formal education" -A.E.

#3 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 14 May 2009 - 06:25 AM

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
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#4 Vini

Vini

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 178 posts
1
Neutral

Posted 15 May 2009 - 01:04 AM

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?

#5 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 15 May 2009 - 02:31 AM

plasmid suffers nicking. fairly common . one nick cause it to lose its supercoiled conformation. happens if stored too long.
Lab + Coffee + Music = Bliss

#6 Vini

Vini

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 178 posts
1
Neutral

Posted 15 May 2009 - 04:28 AM

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....

#7 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 15 May 2009 - 11:49 PM

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!
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#8 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 16 May 2009 - 12:13 AM

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.
Lab + Coffee + Music = Bliss

#9 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 16 May 2009 - 01:15 AM

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!
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#10 WHR

WHR

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 49 posts
0
Neutral

Posted 16 May 2009 - 02:02 AM

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

#11 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 16 May 2009 - 03:06 PM

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.
Lab + Coffee + Music = Bliss

#12 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 17 May 2009 - 05:34 AM

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? :(
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#13 ram

ram

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
0
Neutral

Posted 19 July 2009 - 04:40 AM

Problem located!
That extra band was coming from the lab-made loading dye; donno how :lol: 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?
:( :o ;)
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#14 Vini

Vini

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 178 posts
1
Neutral

Posted 21 July 2009 - 10:00 PM

Problem located!
That extra band was coming from the lab-made loading dye; donno how :D 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?
:( :) :D


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!




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.