Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum > Real-Time PCR and Quantitative PCR Forum
Register Search Today's Posts Mark Forums Read

Real-Time PCR and Quantitative PCR Forum Real-Time PCR and Quantitative PCR Forum


High SYBR green fluorescence at the beggining of the pcr

High SYBR green fluorescence at the beggining of the pcr - Real-Time PCR and Quantitative PCR Forum

High SYBR green fluorescence at the beggining of the pcr - Real-Time PCR and Quantitative PCR Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 01-15-2009, 04:32 PM
Pipette Filler
Points: 77, Level: 1 Points: 77, Level: 1 Points: 77, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jan 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default High SYBR green fluorescence at the beggining of the pcr



Hi,
I work with a native tobacco species, and Im having some trouble with my RT-PCR. I use SYBR green, and Im seeing high fluorescence at the firsts cycles, then, a rise near the Ct and the normal declination during the melting curve stage. The amplification plot seems to be ok and the dissociation plot too. The only problem seems to be the fluorescence plot, its like if I have a lot of dsDNA at the begginig, but Ive run my RT in a gel and there is not any band of genomic DNA. Does anyone know what could be? Idont know if I can trust in the Ct values, having a basal fluorescence too high like this.
Best regards
Reply With Quote
  #2  
Old 01-16-2009, 09:33 PM
danfive's Avatar
Nobel Laureate
Points: 8,612, Level: 64 Points: 8,612, Level: 64 Points: 8,612, Level: 64
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2007
Location: Houston TX
Posts: 969
Thanks: 12
Thanked 184 Times in 154 Posts
Default Re: High SYBR green fluorescence at the beggining of the pcr

If background flourescence is the only problem you may want to 1. prep your reaction on ice, and then transfer them to machine, 2. Incubate rxns (do nothing 4-5min) in dark, easily done within the thermocycle before beginning thermocycle program, 3. cover your reaction tubes from light during preparation.
Reply With Quote
Reply

Tags
beggining , fluorescence , green , high , pcr , sybr


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
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
>>>> LOWER CHOLESTEROL <<<< Janis Stroud Forum Physik 0 09-22-2009 04:39 PM
Sybr Green II and denaturing agarose gels heliozoan RNA Techniques Forum 2 05-11-2008 05:23 AM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM


All times are GMT. The time now is 07:39 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 0.15513 seconds with 16 queries