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


Two standards. Two curves.

Two standards. Two curves. - Real-Time PCR and Quantitative PCR Forum

Two standards. Two curves. - Real-Time PCR and Quantitative PCR Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 05-02-2013, 04:48 AM
Pipette Filler
Points: 97, Level: 1 Points: 97, Level: 1 Points: 97, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2013
Location: Pittsburgh, PA
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Two standards. Two curves.



I'm sorry if the answer to this is something obvious that I'm missing - I am quite new to this and have been trying to figure this out for 2 weeks now.

The Problem:
I am using a standard taqman reaction to amplify viral genomes from dna isolated from mouse tissues (Qiagen DNeasy kit), and am getting very nice and consistent data within experimental groups, standardized to histone control. I am, however, getting very different calculated copies depending on which set of standards I am comparing to. I am amplifying a specific viral gene that has one copy/genome. One set of standards consists of this gene inserted into a small plasmid (2.5kb). The other standard is a Bacterial artificial chromosome (BAC) and contains the entire viral genome (>100kb). The purity of my standard DNA seems OK. However, I get very different results from these standards (1.5-2 logs difference) with the smaller plasmid giving me higher genome copy reads in my samples. The dilutions and calculated copies have been checked and rechecked. The standards both give fantastic curves, but are very far apart. The most obvious answer is that they aren't at the concentration I think they are, or aren't diluted properly, but I have checked and rechecked this. What gives?
Reply With Quote
  #2  
Old 05-02-2013, 03:39 PM
luisillo's Avatar
M.D/Ph.D
Points: 4,149, Level: 43 Points: 4,149, Level: 43 Points: 4,149, Level: 43
Activity: 33% Activity: 33% Activity: 33%
 
Join Date: Jul 2010
Location: Mexico
Posts: 354
Thanks: 20
Thanked 98 Times in 90 Posts
Default Re: Two standards. Two curves.

It is because the plasmid is smaller than the BAC, so in the same DNA concentration you will have a larger quantity of copies of your gene in the plasmid than in the BAC. This will ultimately lead to a larger quantity of copies of your amplicon in the plasmid standard. However, I don't know if this the difference can be as big as you describe (1.5-2 logs), but, as I stated, the plasmid yield will be bigger than the BAC yield.
Reply With Quote
  #3  
Old 05-02-2013, 08:36 PM
Pipette Filler
Points: 97, Level: 1 Points: 97, Level: 1 Points: 97, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2013
Location: Pittsburgh, PA
Posts: 3
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Two standards. Two curves.

So I keep thinking that it must be something like this, but I have calculated the copy numbers correctly based on the size of the plasmids being used, both by hand, and using online tools. I'm thinking there may be contaminating DNA in one of my plasmids, but the both look fine cut vs. uncut on an agarose gel.

Last edited by KaptanOblivious; 05-02-2013 at 08:38 PM.
Reply With Quote
Reply

Tags
curves , qpcr , real time pcr , standards , viral


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
No success generating standard curves... donchoi Real-Time PCR and Quantitative PCR Forum 1 11-24-2008 10:01 PM
FW: [Microbiology] Re: Question on a MacFarland Standards scientifica,inc. Microbiology Forum 0 10-11-2008 10:35 PM
Made to order standards for ICP,ICP/MS and ionic chromatography spectracer Forum Chemia 0 02-25-2007 03:01 AM
Made to order standards for ICP,ICP/MS and ionic chromatography spectracer Forum Chimica 0 02-25-2007 02:57 AM


All times are GMT. The time now is 01:06 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 0.12227 seconds with 16 queries