SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
miseq run troubleshoot and advice on nextera xt library normalization sebl Illumina/Solexa 5 08-20-2015 07:46 AM
Maximum Sample per MiSeq Run Nikvailo Illumina/Solexa 7 03-17-2015 07:00 AM
Volume of algae culture sample required for cDNA library construction angstyle4 Sample Prep / Library Generation 0 05-31-2013 01:19 PM
Can You mix indexed with un-indexed library on MiSeq run ? memento Illumina/Solexa 2 01-22-2013 11:00 AM
Covaris sample volume amed1552 Sample Prep / Library Generation 5 12-05-2012 07:54 AM

Reply
 
Thread Tools
Old 10-22-2015, 07:38 AM   #1
JBKri
Member
 
Location: Heraklion, Greece

Join Date: Jan 2014
Posts: 77
Default MiSeq sample volume, recover leftover library after run?

A MiSeq run needs 600 ul of denatured library. However, about 415 ul of library remains after the run has completed (measured after a v3-600 run). Would it be possible to load a smaller volume? If used in combination with the NextSeq denaturation protocol, a smaller volume would enable a higher cluster density from low-yield samples. This would only work if the sipper goes deep enough into the tube containing the library.

Another (crazy?) thought is to recover the leftover, denatured library, and re-use it in another run if needed, unless there is some sort of backflow or mixing from other wells back into the sample well. Again this only makes sense for low-yield, precious libraries. Any thoughts?

An additional thought; PCR-free library protocols are generally (always?) less biased, but require large amounts of input DNA. The approach with NextSeq denaturation, and possibly less dilution before loading into the cassette, could potentially generate usable libraries from smaller starting amounts of DNA. That seems like a better solution than adding a PCR enrichment step as I discussed here.

Jon

Last edited by JBKri; 10-22-2015 at 08:38 AM.
JBKri is offline   Reply With Quote
Old 10-22-2015, 10:23 AM   #2
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 184
Default

Almost all of that volume is water from the template rinse.
kcchan is offline   Reply With Quote
Old 10-22-2015, 10:51 AM   #3
JBKri
Member
 
Location: Heraklion, Greece

Join Date: Jan 2014
Posts: 77
Default

Quote:
Originally Posted by kcchan View Post
Almost all of that volume is water from the template rinse.
Ah, I see. So my idea wouldn't work. Thanks!
JBKri 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 03:12 AM.


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