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 amplification or non specific band

no amplification or non specific band - PCR - Polymerase Chain Reaction Forum

no amplification or non specific band - 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 03-02-2007, 11:01 PM
Pipette Filler
Points: 1,428, Level: 22 Points: 1,428, Level: 22 Points: 1,428, Level: 22
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default no amplification or non specific band



Hi,
I have a problem abouth my pcr amplification. Some samples (cDNA) which are normally working in the different region, are not working in my protocols but the other samples are working in this protocol. When I changed anneling temperature (-1C), I have seen non specific band at this samples. What should I do now?
Thanks
Reply With Quote
  #2  
Old 03-06-2007, 02:55 AM
moleculardude's Avatar
M.D/Ph.D
 
Join Date: Jul 2006
Location: Melbourne Australia
Posts: 423
Thanks: 5
Thanked 10 Times in 8 Posts
Default Re: no amplification or non specific band

Hello Yucel,
welcome to the forums!
Could you be a bit more specifc. Are the cDNAs all extracted from the same source? If they are not, then it may be expected that some samples will have higher expression than others.

Also if you are preparing the cDNAs, maybe you have prepared some cDNAs different than others. The salt concentration, inhibitory factors or even buffer concentrations may affect the ability of the PCR reaction to occur.

I would suggest checking all your cDNAs first by UV spec (and loading them on a gel) to check quality and quantity.
If they all seem ok, I would only then proceed to PCR. If they are all ok, yet you are still getting problems please post back here.

Cheers!
Reply With Quote
  #3  
Old 03-19-2007, 03:47 PM
Pipette Filler
Points: 1,428, Level: 22 Points: 1,428, Level: 22 Points: 1,428, Level: 22
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: no amplification or non specific band

Hi,
I have cDNAs which are isolated same protocol at the same time. My samples od is 400-500ul. And I have 4 pcr.
My first problem; One of my sample is working for examples in 1 pcr not 2 but I know all my pcr protocols are ok.
Second problem; At the same protocols some pcr products have nonspesifik band?? But not at the others!!
Thans again
YĆ¼cel
Reply With Quote
  #4  
Old 03-24-2007, 03:54 PM
Pipette Filler
Points: 1,355, Level: 21 Points: 1,355, Level: 21 Points: 1,355, Level: 21
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2007
Posts: 9
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: no amplification or non specific band

Hi all,

I order to figure out what is wrong in your reactions, you need to be a little bit more detailed on
1. What organisms are you extraction from
2. How do you do reverse transcription
3. What primers are u using
4. What are the primer and DNA concentrations
5. What are the PCR conditions

In general, it is helpful to measure DNA concentration but that gives you little info about the Quality of your DNA. If the material you are extracting is old, then your DNA might not be the best for amplification. Hence you get some samples with good PCR bands and others just won't work. Also, if your DNA concentration is too high, you will get a smear rather than a nice band.
The same is true for the primer concentration (more than one band when using too much)
So, in order to really tell what went wrong, we need more info!!!
Reply With Quote
Reply

Tags
amplification , band , 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

Similar Threads
Thread Thread Starter Forum Replies Last Post
nice tight and dark non specific band Tfal Western Blot Forum 0 05-15-2009 04:52 AM
Calculations for specific activity Morales, Victor Protein Forum 0 10-02-2008 01:17 PM
non specific amplification in clone fragment using primer muhammad yasir Protocols and Methods Forum 1 12-21-2006 01:14 PM
non specific amplification in clone fragment using primer aganist vector WS Protocols and Methods Forum 0 12-16-2006 05:54 PM
combining Conduction Band theory with Electronegativity as one phenomenon Archimedes Plutonium Chemistry Forum 2 10-12-2003 06:25 PM


All times are GMT. The time now is 03:59 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 0.13568 seconds with 16 queries