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.


No Bands After PCR

No Bands After PCR - PCR - Polymerase Chain Reaction Forum

No Bands After PCR - 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 02-19-2008, 06:11 PM
Pipette Filler
Points: 732, Level: 14 Points: 732, Level: 14 Points: 732, Level: 14
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2008
Posts: 5
Thanks: 0
Thanked 0 Times in 0 Posts
Question No Bands After PCR



Hi,
i am not getting any pcr bands on agarose gel electrophoresis!!

what should i do??? i have tried all temperatures and optimization....
Reply With Quote
  #2  
Old 02-19-2008, 07:41 PM
Graduate Student
Points: 2,923, Level: 35 Points: 2,923, Level: 35 Points: 2,923, Level: 35
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2006
Location: Ithaca, NY
Posts: 145
Thanks: 0
Thanked 17 Times in 12 Posts
Default Re: No Bands After PCR

Well, I'm not sure what "optimization" you've tried so here's some things I'd try:

1) PCR additives: DMSO, betaine
2) Less template: see if there's a PCR inhibitor in the template DNA (knowing what kind of DNA you're starting with would help troubleshooting)
3) Positive control: Do you have a known template your primers will amplify? Or other primers that you know amplify your template? Or some PCR product you can amplify to make sure the thermocycler is working properly each run?
4) New primers: sometimes primer sequences just don't work -- did you check for hairpin formation, homo- and hetero- dimerization, matched Tms, etc.? Or perhaps the primer synthesis didn't work properly. Order some new primers and try all combinations.
5) Make sure the gel is running correctly -- does your size ladder migrate and stain properly?
Reply With Quote
  #3  
Old 02-20-2008, 09:40 AM
Pipette Filler
Points: 1,804, Level: 26 Points: 1,804, Level: 26 Points: 1,804, Level: 26
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2008
Posts: 29
Thanks: 0
Thanked 2 Times in 2 Posts
Default Re: No Bands After PCR

If the suggestions by kmunson dont work Change the water you are using!!!! may be that would work
Reply With Quote
  #4  
Old 04-18-2008, 05:18 PM
Pipette Filler
Points: 568, Level: 11 Points: 568, Level: 11 Points: 568, Level: 11
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2008
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: No Bands After PCR

plz post-up what did you do and what optimization have you tried so we'll be more helpful as the problem of not getting any bands might belong to punch or reasons. so plz put up whatever protocols you've used before so we can exclude unneccessary criteria.

thx
Reply With Quote
  #5  
Old 04-23-2008, 05:01 PM
Pipette Filler
Points: 639, Level: 12 Points: 639, Level: 12 Points: 639, Level: 12
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Apr 2008
Posts: 13
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: No Bands After PCR

are you using an oil overlay?

If not, make sure the heated lid is working and that it fits down snug on top of your PCR rxn tubes. This was giving me trouble on my new thermocycler, I wasn't putting the tubes in correctly and the top wasn't snug.
Reply With Quote
  #6  
Old 05-30-2008, 11:57 AM
Pipette Filler
Points: 533, Level: 10 Points: 533, Level: 10 Points: 533, Level: 10
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2008
Posts: 7
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: No Bands After PCR

Maybe forgotten to add one of the pivotal PCR ingredients??? Forgetting to add Taq Polymerase always give no yield
Reply With Quote
  #7  
Old 01-24-2009, 10:55 AM
NATHANALANPATRICKROGERS's Avatar
Summer Student
Points: 306, Level: 6 Points: 306, Level: 6 Points: 306, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Location: Maroochydore
Posts: 73
Thanks: 4
Thanked 13 Times in 11 Posts
Default Re: No Bands After PCR

Quote:
Originally Posted by labratty View Post
Hi,
i am not getting any pcr bands on agarose gel electrophoresis!!

what should i do??? i have tried all temperatures and optimization....
Your initial DNA could in fact be nothing more than glorified moon dust
Reply With Quote
  #8  
Old 01-28-2009, 02:56 PM
Pipette Filler
Points: 3,744, Level: 40 Points: 3,744, Level: 40 Points: 3,744, Level: 40
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 27
Thanks: 1
Thanked 2 Times in 2 Posts
Default Re: No Bands After PCR

I wasn't getting any bands with my PCRs until I re-amped it. I took 1ul of the PCR product and used it as template in a new PCR mix and that gave me product. I was adding primers to a total DNA extraction and my gene of interest was expressed at a very low level so any amplification that I did get was still too small to visualise on a gel.
Reply With Quote
The Following User Says Thank You to cat1082 For This Useful Post:
admin (01-28-2009)
  #9  
Old 01-29-2009, 08:13 AM
Banned
Points: 27, Level: 1 Points: 27, Level: 1 Points: 27, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 6
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: No Bands After PCR

Hey,
from my experience most of the time it is a problem related to the primers.You should check for dimerization errors or hairpin formation. If all this doesn't work, just order some new primers with different combinations as primers aren't that expensive you shouldn't hesitate too long.

cheers
Heidi
Reply With Quote
Reply

Tags
bands , pcr


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
negative bands problem SallyG SDS-PAGE Gel Electrophoresis Forum 4 07-06-2010 08:03 PM
Increase Exposure, Bands Disappear?? Mediatorx Western Blot Forum 3 07-01-2008 09:18 PM
Help Migrating bands camherst Western Blot Forum 1 01-26-2008 02:48 PM
Artifactual double bands Natalia Oliveira Protocols and Methods Forum 1 09-08-2007 11:28 AM
cloning 16S rDNA TRFLP bands _wallnut2@excite.com Protocols and Methods Forum 0 04-27-2005 10:53 PM


All times are GMT. The time now is 03:25 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 0.15922 seconds with 16 queries