SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
Pooling multiplexed libraries after ligation, before PCR ScottC Illumina/Solexa 5 05-07-2016 02:10 PM
Purification of Nextera libraries for use on the MiSeq vieiraL General 5 03-27-2013 05:26 AM
Genomic DNA libraries using Nextera creeves Illumina/Solexa 3 03-02-2013 12:03 PM
qPCR on Nextera XT Libraries kcchan Sample Prep / Library Generation 3 08-30-2012 08:29 AM
Indexed libraries best pooling strategy spdinesh Sample Prep / Library Generation 3 02-08-2012 12:21 AM

Reply
 
Thread Tools
Old 09-17-2013, 07:00 AM   #1
kmkocot
Member
 
Location: Alabama

Join Date: Jun 2009
Posts: 48
Question Pooling and diluting Nextera libraries

Hi all,

I made some Nextera libraries last week and wanted to pool 8 of them for sequencing. Here's the Qubit concentration and volume containing 2 nM for each given the equation 1ng/ul = 3nM (which should be accurate given my size distributions).

Qubit result (ng/uL) / Volume containing 2 nM
0.073 / 9.13
0.904 / 0.74
9.01 / 0.07
2.34 / 0.28
1.13 / 0.59
1.43 / 0.47
7.24 / 0.09
0.26 / 2.56

In order to pool the libraries, I was going to take the amount that equals 2 nM of library for each, pool that (in this case resulting in a total volume of 22.39 microliters) and use 5 microliters of that for denaturation and dilution (according to the protocol that begins on the bottom of page 9 of this document:
http://supportres.illumina.com/docum...15039740-c.pdf).

Does this sound logical? I realize this is basically a high-school chemistry question but I'm a bit mathophobic and want to be sure I'm doing this correctly.

Thanks!
Kevin
kmkocot is offline   Reply With Quote
Reply

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 07:42 PM.


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