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
- - - - -

LacZ PCR genotyping -ve control contamination


  • Please log in to reply
3 replies to this topic

#1 Dave_Kub_11

Dave_Kub_11

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 13 February 2010 - 10:03 AM

Hello everyone. I am new to this forum and wonder if someone could help me with a rather annoying problem as I have tried to troubleshoot it the best I can!!

I have been getting a band popping up in my negative controls in my genotyping for the lacZ transgene. The contaminant band is the same size as that of the LacZ (~315bp)

I used fresh PCR reagents, fresh dilutions of primers, water, pipette tips etc. and I am still getting this contamination. I have done this several times now.

I have ordered new primers as I suspect the contamination might be there.

If anyone has any experience of having this problem, particularly with lacZ (which I am told is rather susceptible to contamination) I will be much appreciated if you could help me out!!!

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,666 posts
396
Excellent

Posted 14 February 2010 - 03:30 PM

You are working off a plasmid that has probably been extracted thousands of times in the lab you are working in, I'm not surprised you have contamination issues, the air will be full of it.

I suggest trying setting your PCR up in another room if possible; somewhere where no plasmids are extracted/bugs grown. If that isn't possible, try keeping a set of pipettes just for setting up PCR, and have a PCR region, where you only set up PCRs.

#3 Dave_Kub_11

Dave_Kub_11

    member

  • Active Members
  • Pip
  • 22 posts
0
Neutral

Posted 15 February 2010 - 10:36 AM

You are working off a plasmid that has probably been extracted thousands of times in the lab you are working in, I'm not surprised you have contamination issues, the air will be full of it.

I suggest trying setting your PCR up in another room if possible; somewhere where no plasmids are extracted/bugs grown. If that isn't possible, try keeping a set of pipettes just for setting up PCR, and have a PCR region, where you only set up PCRs.



I set up PCR in another room as you suggested and I am still getting this contamination occurring. I even cleaned my PCR workstation quite thoroughly and it didn't make any difference. I used fresh primers that arrived today and I still got the contamination.

I wonder if DMSO might be causing this (although I have used two different bottles to check if that is the cause of the contamination, no luck)

#4 lab rat

lab rat

    Why does a science forum not have pictures of mice and rats?

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 245 posts
7
Neutral

Posted 15 February 2010 - 09:23 PM

Maybe your pipettes are contaminated. Are you using barrier tips?
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.




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

©1999-2013 Protocol Online, All rights reserved.