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
- - - - -

Gel Electrophoresis Smearing Problems


  • Please log in to reply
4 replies to this topic

#1 Shauki

Shauki

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 02 April 2011 - 04:06 AM

Hi guys!..

This is my first ever post in this forum!
I have few problems that occured when I doing my mini molecular biology project. It was so stressful and mind-blowing to troubleshoot the problems and leads to difficulties for me to make a disscussion on my project.
Problems:
I encountered heavy smearing problems after I did gel electrophoresis. I have repeated it several times but smearing still occurs. Electrophoresis was done on 1% agarose gel. cDNAs that I used are cDNAs of plant extracts (hibiscus rosa sinensis) treated-breast cancer cell lines.
The image can be seen at this link -> http://uploadpic.org...MpFcxIJiI2d.jpg
Notes that ACP reffered to annealing control primer.(Seegene's DEG kit)
So, what exactly the main causes of the smearing and what is the other factors that may involved in gel electrophoresis? I tried to perform further optimization but due to time limitation, I can't.

Thanks..

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,709 posts
123
Excellent

Posted 04 April 2011 - 10:06 AM

smearing is caused by overloading and by multiple species.

are you preparing the cdna from a specific mrna or are you making the cdna from all of the mrna?
talent does what it can
genius does what it must
i do what i get paid to do

#3 chimpsarehungry

chimpsarehungry

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 04 April 2011 - 11:26 AM

Sometimes the smearing is caused by your DNA in the process of degradation. As in, it is cut up into all kinds of pieces by DNases. Some ways to fix it include eluting your DNA in T.E. instead of water, or using a new stock of T.E. in case contaminated, or keeping your DNA at colder temperatures.

#4 Shauki

Shauki

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 04 April 2011 - 07:59 PM

@mdfenko : cDNA is actually prepared by conducting reverse trancription of the extracted RNA from the extracted cell lines..

@chimpsarehungry : thanks..i also thought that my smearing problems is largely due to degradation of DNA..

#5 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,709 posts
123
Excellent

Posted 05 April 2011 - 12:23 PM

are you preparing cdna with a specific primer? if not then smearing may be more due to multiple species of cdna than to degradation.

if the smearing is below the band then it may be more due to degradation. if it is also above the band then it may be more due to overload and/or multiple species (your picture looks like smearing above and below bands).
talent does what it can
genius does what it must
i do what i get paid to do




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

©1999-2013 Protocol Online, All rights reserved.