Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
Nextera XT library troubleshooting sebl Sample Prep / Library Generation 0 11-03-2014 06:46 AM
Qubit library quantitation variations Smriti Sample Prep / Library Generation 5 11-02-2013 10:57 AM
DNA quantitation & library preparation problem charlescoldroom Illumina/Solexa 7 06-21-2012 06:05 AM
Rapid Library Library Quantitation using concentration not RFU GraemeFox 454 Pyrosequencing 9 10-21-2011 12:27 AM
Library quantitation discrepancy genlyai Illumina/Solexa 1 04-15-2011 08:24 AM

Thread Tools
Old 09-14-2018, 02:19 AM   #1
Junior Member
Location: Scotland

Join Date: Oct 2015
Posts: 2
Question Quantitation of Nextera XT library

I have searched the forum without success so apologies in advance if I have missed this elsewhere.

We are sequencing pooled Nextera XT preps of bacterial genomes, using the bead normalisation. Until now we have not carried out any post-normalisation quantitation and have been reasonably happy with clustering on the MiSeq V3 of between 1000K and 1600K/mm2

Recently, we have had a couple of failed runs with low clustering (<300K), possibly as a result of changes in staffing. We would like to use the Qubit ssDNA kit to quantify the pooled libraries. Does anyone have experience/suggestions/protocols?
Cdiff is offline   Reply With Quote
Old 09-14-2018, 04:03 AM   #2
Junior Member
Location: New York

Join Date: Aug 2017
Posts: 7

I think you'll need the dsDNA assay kit since these are libraries. It's fairly straightforward and you can just follow the manufacturer's protocol. Nothing particularly special needs to be done for library quantification.
Buckethead84 is offline   Reply With Quote
Old 09-14-2018, 04:25 AM   #3
Junior Member
Location: Scotland

Join Date: Oct 2015
Posts: 2

We use the HS dsDNA kit for the Nextera XT input sample. But to measure the pooled normalised library, I think we need the ssDNA kit as the NaOH elution produces ss. Or am I wrong here?
Cdiff is offline   Reply With Quote
Old 09-14-2018, 04:51 PM   #4
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,199

Link to Qubit ssDNA manual below. You might need to use higher volume of pool for quantification. Start with 5 ul and if signal is below detection it can be increased up to 20 ul.

You will need to set up a small volume PCR reaction to estimate pool size for calculating molar concentration. Primers should be complementary to P5 and P7 flow cell binding motives such as primer cocktail form a TruSeq PCR or KAPA qPCR kit.
nucacidhunter is offline   Reply With Quote

library concentration, nextera xt, qubit

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 10:28 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO