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.


primer dimer and non specific bands

primer dimer and non specific bands - PCR - Polymerase Chain Reaction Forum

primer dimer and non specific bands - 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 05-29-2010, 10:46 AM
Pipette Filler
Points: 14, Level: 1 Points: 14, Level: 1 Points: 14, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default primer dimer and non specific bands



I'm doing nested pcr using Bioron mastermix beads 50μl reaction and according to the protocol I follow the first round primers are used in 0.2μM concentration,The second round primers are in 2μM conc.Not only primer dimer bands of 50bps appear with all samples but other bands appear at about 100bps but not with all samples .My target gene is 322bp which appeared once.could primer dimer prevent target amplification in other samples and what are those non specific bands.thanks
Attached Images
File Type: jpg 26052010(006).jpg (96.5 KB, 11 views)
Reply With Quote
  #2  
Old 06-01-2010, 03:52 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: primer dimer and non specific bands

Try checking the sequence of the primers for dimers, rise the annealing temperature to make the pcr more specific and if you still have problems, try to just excise from the gel the band you want to work with.

And yes, primer dimers could prevent target amplification because the primers are "consumed" by generating dimers and then they won't be useful for the polymerase
Reply With Quote
  #3  
Old 06-08-2010, 02:33 PM
Pipette Filler
Points: 283, Level: 5 Points: 283, Level: 5 Points: 283, Level: 5
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2010
Posts: 4
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: primer dimer and non specific bands

I've checked my primer with DNA calculator previously, the result was none primer dimer. But when I use it for PCR at the concentration of 20microM there are always none specific bands. After I changed the concentration to 1microM (based on the TaqPol protocol) unspecific band dissapears. I think the final concentration of the primer in the PCR mix also rules..
Reply With Quote
  #4  
Old 06-07-2011, 10:41 PM
Pipette Filler
Points: 62, Level: 1 Points: 62, Level: 1 Points: 62, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jun 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: primer dimer and non specific bands

1) i performed a pcr using two different primers on mushroom fruitbody and sclerotium of one of the mushrooms. the genus specific primer designed gave one band for all samples but the ITS 1 (forward) and ITS 4 (reverse) gave one band for fruitbody and two bands for sclerotium (one faint band on top of a major band). what is the cause?

2) How do i use this result to identify my samples?
Reply With Quote
Reply

Tags
bands , dimer , primer , specific


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


All times are GMT. The time now is 03:03 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 0.15726 seconds with 16 queries