SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
Size-selection of TruSeq DNA library ageneheo Sample Prep / Library Generation 2 12-03-2012 08:15 AM
Size-selection with ScriptSeq v2 RNAseq Library? Jean Sample Prep / Library Generation 0 11-14-2012 06:37 AM
Size selection of ChIP-Seq library OptimusBrien Sample Prep / Library Generation 7 02-22-2012 12:18 PM
Library size selection for bacterial RNAseq whitestein General 2 08-19-2011 05:36 AM
Paired End Library Size Selection GeCKo 454 Pyrosequencing 2 11-16-2010 07:06 AM

Reply
 
Thread Tools
Old 07-25-2016, 11:54 AM   #1
LacquerHead
Member
 
Location: New York

Join Date: Nov 2015
Posts: 31
Default Library size selection

Hi everyone,

Want to size select some Nextera libraries and wondering about everyones preferred method? I have tried Blue Pippin but the cassette % size constraints make this difficult. Moving to trying with gel and want to get an idea of what % of what kind of agarose (e.g. MetaPhor) people are using and running conditions? I am trying to select 150-800bp approximately. Thank you for your feedback!

LH
LacquerHead is offline   Reply With Quote
Old 07-25-2016, 12:15 PM   #2
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 64
Default

Hello Lacquerhead,

I have done a lot of gel size selections with 2% agarose gels which worked well. I used low-melt agarose, and don't think the variety matters too much. If you are selecting down to 150bp you will want to make sure that you let your sample migrate long enough to separate fully from any potential dimers that will be ~120bp.
jdk787 is offline   Reply With Quote
Old 07-25-2016, 01:01 PM   #3
LacquerHead
Member
 
Location: New York

Join Date: Nov 2015
Posts: 31
Default

Don't have anything at 120 in these samples, only around 50bp so shouldn't be a problem. Which kit do you use? Ive heard a suggestion to use regular gel extraction kit but with the MinElute columns.
LacquerHead is offline   Reply With Quote
Old 07-25-2016, 01:08 PM   #4
LacquerHead
Member
 
Location: New York

Join Date: Nov 2015
Posts: 31
Default Gel stain

Another question is how do you stain the gel, EtBr or something else like SYBR Gold?
LacquerHead is offline   Reply With Quote
Old 07-25-2016, 01:21 PM   #5
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 64
Default

I work at Bioo Scientific, so I just use our reagents.
http://www.biooscientific.com/Illumi...Kit-Components but any gel any gel selection kit should be fine.

Both Ethidium Bromide and SYBR Gold will work.
jdk787 is offline   Reply With Quote
Old 07-25-2016, 01:39 PM   #6
LacquerHead
Member
 
Location: New York

Join Date: Nov 2015
Posts: 31
Default

How is the efficiency of the kit in terms of ng recovered? In this lot range of 150-800bp? Thanks.



Quote:
Originally Posted by jdk787 View Post
I work at Bioo Scientific, so I just use our reagents.
http://www.biooscientific.com/Illumi...Kit-Components but any gel any gel selection kit should be fine.

Both Ethidium Bromide and SYBR Gold will work.
LacquerHead is offline   Reply With Quote
Old 07-25-2016, 04:39 PM   #7
LacquerHead
Member
 
Location: New York

Join Date: Nov 2015
Posts: 31
Default

Does anyone have good protocols for removing fragments larger than 800-1000bp with Ampure XP beads?
LacquerHead is offline   Reply With Quote
Old 07-26-2016, 03:30 AM   #8
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,226
Default

I wonder why you want to size select a Nextera library in 180-800 bp range. If you sequence a library at this size range 80% of reads will be from fragments below 500 bp and hardly any reads from fragments larger than 800 bp.

I do not have a validate protocol but you would need to do a double SPRI. In principal, add 0.48X bead and take the supernatant then add more beads to make the bead ratio 0.8X. I would suggest doing this with sheared DNA if it is available to make sure that your batch of AMPure beads gives desired results.
nucacidhunter is offline   Reply With Quote
Old 07-26-2016, 08:30 AM   #9
jdk787
josh kinman
 
Location: Austin

Join Date: Apr 2014
Posts: 64
Default

Quote:
Originally Posted by LacquerHead View Post
How is the efficiency of the kit in terms of ng recovered? In this lot range of 150-800bp? Thanks.
The gel selections that I have done have been before amplification, so I don't have a lot of data on recovery, but would expect >60% recovery.

A bead based size selection should have a higher yield than using a gel, and I agree with nucacidhunter in regards to the upper ratio and using fragmented DNA to validate beforehand.
jdk787 is offline   Reply With Quote
Old 07-26-2016, 10:33 AM   #10
Markiyan
Senior Member
 
Location: Cambridge

Join Date: Sep 2010
Posts: 115
Lightbulb Do a deplition beads bind @ ~0.5X, to bind large fragments.

Quote:
Originally Posted by LacquerHead View Post
Does anyone have good protocols for removing fragments larger than 800-1000bp with Ampure XP beads?
Do a large fragment depletion beads bind at 0.6X - 0.45X and take the supernatant forward (add more beads to supernatant, to capture the smaller fragments - like 1.5X - 2X).

QC by bioanalyser, adjust ratios as needed.
Markiyan 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 08:12 PM.


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