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.


negative control gives band same as positive control

negative control gives band same as positive control - PCR - Polymerase Chain Reaction Forum

negative control gives band same as positive control - 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 04-16-2010, 06:49 AM
Pipette Filler
Points: 39, Level: 1 Points: 39, Level: 1 Points: 39, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Question negative control gives band same as positive control



I run PCR with RNA (untreated with DNAse) to see the if there was integration of transgene to genome. But my PCR negative controls showed band same size as my positive control and it is as intense as my positive control. I ran another PCR to confirm that I dont accidently change my negative control with sample but the band still there for the second time. if it cause by master mix contamination then it is suppose to be that all my lane have the same band right. or it is possible that my master mix contaminate. please help me
Reply With Quote
  #2  
Old 04-16-2010, 07:11 AM
Pipette Filler
Points: 1,961, Level: 27 Points: 1,961, Level: 27 Points: 1,961, Level: 27
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2010
Location: Norway
Posts: 22
Thanks: 0
Thanked 4 Times in 4 Posts
Default Re: negative control gives band same as positive control

If a negative control is positive, I would think it's some kind of contamination.

If your mastermix is contaminated, then all of your bands will have the same band if all of your samples are made with the same mastermix.

But it's also possible that it's only your negative control in itself that is contaminated
Reply With Quote
  #3  
Old 04-16-2010, 09:37 AM
Pipette Filler
Points: 39, Level: 1 Points: 39, Level: 1 Points: 39, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2010
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: negative control gives band same as positive control

Quote:
Originally Posted by Aurora Borealis View Post
If a negative control is positive, I would think it's some kind of contamination.

If your mastermix is contaminated, then all of your bands will have the same band if all of your samples are made with the same mastermix.

But it's also possible that it's only your negative control in itself that is contaminated
thanx Aurora

yeah now that you say so, I also don't think my master mix was contaminated. my supervisor ask me to re run my pcr with his mastermix. hope this time my negative control doesn't give unwanted band
Reply With Quote
  #4  
Old 04-19-2010, 05:03 PM
Volunteer
Points: 1,375, Level: 21 Points: 1,375, Level: 21 Points: 1,375, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2009
Posts: 48
Thanks: 10
Thanked 9 Times in 8 Posts
Default Re: negative control gives band same as positive control

Make sure you run a lane with no DNA or RNA--replace your template with plain water. This will tell you if your band is produced by contamination in the master mix, or if there's some endogenous sequence or contaminating sequence in your negative control cDNA that is amplifying.
Reply With Quote
  #5  
Old 04-26-2010, 06:03 PM
Pipette Filler
Points: 995, Level: 17 Points: 995, Level: 17 Points: 995, Level: 17
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: negative control gives band same as positive control

Hi adiza!With my little experience of pcr, your negative control might have been contaminated from previous pcr amplification products (amplicons) or as a result of cross contamination. So i suggest that you repeat the whole process after thorough decontamination of the workbenches and equipments.
Reply With Quote
Reply

Tags
band , control , negative , positive


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
Positive dot blot, negative WB durante Western Blot Forum 1 04-22-2010 09:07 AM
negative control in Q-PCR how to analyze results? gozde06 Real-Time PCR and Quantitative PCR Forum 0 02-27-2010 09:39 PM
dsRNAi positive control Man Mohan Drosophila Forum 0 10-23-2004 04:17 PM
Positive and Negative Control agy lee Microbiology Forum 1 12-18-2003 07:39 PM


All times are GMT. The time now is 02:55 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13853 seconds with 16 queries