Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
qubit/picogreen seqgirl123 Sample Prep / Library Generation 4 11-02-2013 07:06 AM
Can you use PicoGreen in a Qubit assay? crader General 2 12-05-2012 10:35 AM
Rapid Library Library Quantitation using concentration not RFU GraemeFox 454 Pyrosequencing 9 10-20-2011 11:27 PM
Quality Control Library using QUBIT... help me! soleulloa Ion Torrent 1 09-01-2011 06:18 AM
Library quantitation discrepancy genlyai Illumina/Solexa 1 04-15-2011 07:24 AM

Thread Tools
Old 11-08-2011, 10:34 AM   #1
Location: California

Join Date: Mar 2011
Posts: 40
Default Qubit library quantitation variations

Hi All,

I am facing lot of variations in my library quantitation using Qubit dsDNA BR assay kit. Lambda DNA stock concentration which is provided by Sigma has a documented concentration of 400ng/ul and Qubit shows above 550ng/ul. What might be the root cause? Is it pipetting error or people tend to get variations from sample to sample?

Please advise!
Smriti is offline   Reply With Quote
Old 12-26-2011, 04:18 PM   #2
Location: New York

Join Date: Dec 2011
Posts: 20

Hi, it could be pipetting errors, but more likely you simply have a bit of batch variation from Sigma so that it's not exactly 400 ng/uL. However, I don't have any experience with these stocks so anyone else can comment if they know.
Gina_P is offline   Reply With Quote
Old 12-27-2011, 09:25 AM   #3
Location: Boulder, CO

Join Date: Sep 2011
Posts: 19


In our experience the Qubit BR assay is less accurate than the HS assay, so try doing a dilution of your DNA and using the HS kit if you have it. Our BR assay usually read higher than the HS, even if the stock DNA concentration fell within the range of both.

One QC thing we've started is doing a spec of your Qubit standards, especially standard 2. We had one kit with problems and found it was almost 20% above the concentration written on the tube.

Also mix your samples extremely well, this can make a huge difference especially if you're only measuring 1ul.

lterhune is offline   Reply With Quote
Old 11-02-2013, 05:41 AM   #4
Senior Member
Location: UK

Join Date: Jul 2013
Posts: 131
Default Qubit (HS, BR)

when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?
mmmm is offline   Reply With Quote
Old 11-02-2013, 09:06 AM   #5
Location: Boston, MA

Join Date: Apr 2012
Posts: 14

When was the last time you ran the standards? If you're not running them every time then you're pretty susceptible to pipetting errors in your Q-reagent/buffer sln.
chevrm is offline   Reply With Quote
Old 11-02-2013, 09:57 AM   #6
Senior Member
Location: UK

Join Date: Jul 2013
Posts: 131

I run the standard everytime and Qubit reads the right conc. for the standard but I got different readings for the same sample (20ng/Ml when HS kit was used) and (80ng/Ml when BR kit was used), have tried on other samples too and get different readings- it is not pipeeting errors so what is the explanation?

Also, would DNA concentration decrease that much following freezing and thawing?
mmmm is offline   Reply With Quote

Thread Tools

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

All times are GMT -8. The time now is 10:15 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO