Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


PCR negative control contamination with bands of a different size!!

PCR negative control contamination with bands of a different size!! - PCR - Polymerase Chain Reaction Forum

PCR negative control contamination with bands of a different size!! - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 06-11-2010, 08:12 PM
Pipette Filler
Points: 727, Level: 14 Points: 727, Level: 14 Points: 727, Level: 14
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Unhappy PCR negative control contamination with bands of a different size!!



Hi all!

I have a thing going on with my PCR! My negative controls, both the mix one and the one when you add the DNA sample, show contamination. But the thing is the band (around 400 bp) is different that the target sequence. I'm typifing Trypanosoma cruzi from bugs' rectal ampoule samples, so I do a series of PCR with different target sequence but this bands keeps showing up!
I guess my two mail questions are:
1-What is going on?!
2-Is this considered contamination? Because, I mean, the band size is different that the positive bands you get if you have any type of T. cruzi...

Thanks a lot!
Reply With Quote
  #2  
Old 06-11-2010, 09:27 PM
Graduate Student
Points: 2,626, Level: 33 Points: 2,626, Level: 33 Points: 2,626, Level: 33
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2010
Location: Santiago, Chile
Posts: 100
Thanks: 1
Thanked 24 Times in 24 Posts
Default Re: PCR negative control contamination with bands of a different size!!

Some questions first:

The "contamination" band you always get is always the same size? It appears in all your PCR?

What's the PCR program you're using for every primer pair? Are all the same or not?

Are all PCR reactions equally on DNA concentration, primers and everything else?

What do you mean by "mix" negative control and "the one when you add the DNA sample" negative control?

This can give us a better clue of what's going on
Reply With Quote
  #3  
Old 06-14-2010, 03:36 PM
Pipette Filler
Points: 727, Level: 14 Points: 727, Level: 14 Points: 727, Level: 14
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: PCR negative control contamination with bands of a different size!!

Hi! thanks for taking an interest!

Yes, the band is always the same size and yes it does appear in more than one PCR. The program varies from primer pair to another.
the DNA concentration is the same for all of them while the primers, Mg, buffer, etc isn't.
Regarding the negative controls, I have two for each round of PCR, both with water instead of DNA template. The first one is while doing the master mix. I put the mix in all the little eppendorfs and then in one of them I put water. Later on I don't add anything else. The other is while adding the DNA samples in the mix. That is in another eppendorf I put water instead of DNA. The water for each control is from a different water stock. The idea of making two negative controls is to test for possible contamination in this two stages.
Well, I hope this clarifies a bit!
Reply With Quote
  #4  
Old 06-15-2010, 02:46 AM
Graduate Student
Points: 2,626, Level: 33 Points: 2,626, Level: 33 Points: 2,626, Level: 33
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2010
Location: Santiago, Chile
Posts: 100
Thanks: 1
Thanked 24 Times in 24 Posts
Default Re: PCR negative control contamination with bands of a different size!!

You said the contamination appears in more than one PCR. I can deduce from what you said that the contaminationa appears only in some PCR (more than one) but not in all your PCR programs. Is that true? Because if it only appears in some PCR, you should check what do those PCR program/mixes have in common. DNA or primer concentration, some other reactive, etc.

I thought it was water that was contaminated, because it's kinda common it cross-contaminates with the target DNA, or the primers, but now you mention you vary the Mg and the buffer, it gives me something else to think, because you can't simply state the water is not contaminated "because the contamination doesn't appear in all the pcr I use this water in" because you're varying many other factors that can be implied.

Check what things share in common those PCR that show the contamination in their negative controls, and this way you can better state where is the problem.
Reply With Quote
The Following User Says Thank You to SebaQ For This Useful Post:
Luli (07-01-2010)
  #5  
Old 07-01-2010, 07:46 PM
Pipette Filler
Points: 727, Level: 14 Points: 727, Level: 14 Points: 727, Level: 14
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: PCR negative control contamination with bands of a different size!!

Yeah, I did some tests and I think it was the sample of water I had taken but also some contamination in the primers.

Thanks!
Reply With Quote
Reply

Tags
bands , contamination , control , negative , pcr , size


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
inconsistent PCR contamination (band in negative control) metabus PCR - Polymerase Chain Reaction Forum 3 06-14-2010 12:53 PM
negative control gives band same as positive control adiza PCR - Polymerase Chain Reaction Forum 4 04-26-2010 06:03 PM
negative control in Q-PCR how to analyze results? gozde06 Real-Time PCR and Quantitative PCR Forum 0 02-27-2010 09:39 PM
GNU units and units.dat; Units of Measurement and Unit Conversion James Redford Physics Forum 0 07-31-2005 12:08 PM
Positive and Negative Control agy lee Microbiology Forum 1 12-18-2003 07:39 PM


All times are GMT. The time now is 12:21 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.23016 seconds with 16 queries