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.


Fecal DNA PCR not working

Fecal DNA PCR not working - PCR - Polymerase Chain Reaction Forum

Fecal DNA PCR not working - 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 01-24-2010, 08:38 AM
Pipette Filler
Points: 54, Level: 1 Points: 54, Level: 1 Points: 54, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2010
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Fecal DNA PCR not working



Hi,
I am doing genotyping of wild ass fecal DNA.The PCR program I use for amplifying genomic DNA for microsatellite genotyping is:
95 deg celsius, 5 min
then 3 cycles of:
15'' @ 95 deg;
30'' @ 60 deg;
30'' @ 72 deg;
then 3 cycles of the same but with 58 deg instead of 60, then 56....48, then 30 cycles of the same @ 48 deg.
This touchdown program was not developed by me, but was paritally working when I joined the lab. A simple "regular" PCR program does not work for these primers.
In most reactions, only the positive control works (blood DNA), while my samples which are fecal DNA, do not work.

What can I do to improve the PCR conditions to make the fecal DNA work?
Reply With Quote
  #2  
Old 01-24-2010, 09:34 PM
Aga's Avatar
Aga Aga is offline
Post-Doc
Points: 2,239, Level: 30 Points: 2,239, Level: 30 Points: 2,239, Level: 30
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 270
Thanks: 25
Thanked 78 Times in 64 Posts
Default Re: Fecal DNA PCR not working

Try to improve your DNA extraction protocol - feces contain many PCR inhibitors and without additional DNA extraction steps PCR doesn't work efficiently, if at all.
Have you checked your DNA purity and amount?
Reply With Quote
Reply

Tags
dna , fecal , pcr , working


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
pENTR1A Transformation not working CambridgeBiochemist Molecular Cloning Forum 1 06-18-2012 06:53 AM
As a bio major, should i be concerned with working with HIV? MCB Science Jobs Forum 1 06-09-2010 09:41 PM
smallest working peptide kukua Peptide Forum 2 02-23-2008 02:42 AM
[Zbrafish] Working schedule for mid- to late- somite stage study Burdine, Rebecca D Zebrafish Forum 0 04-26-2007 07:49 PM
working for the man lyrics pj harvey Physics Forum 0 06-28-2005 12:23 PM


All times are GMT. The time now is 04:54 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 0.11733 seconds with 16 queries