SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Sequence of Nextera transposon Mikemike107 Illumina/Solexa 3 05-26-2016 07:47 AM
How to blast a transposon sequence to a MiSeq transposon library helelein Bioinformatics 5 07-22-2014 09:52 AM
transposon/insertion sequencing techniques lvenga Genomic Resequencing 1 05-30-2013 12:17 PM
Chimera generation across transposon: how to avoid? pag Sample Prep / Library Generation 2 10-11-2012 04:44 AM
Sequence transposon flanking region Akira Sample Prep / Library Generation 8 03-18-2012 06:53 AM

Reply
 
Thread Tools
Old 05-25-2019, 08:31 AM   #1
sobbewoscht
Junior Member
 
Location: DE

Join Date: May 2019
Posts: 1
Default Check transposon activity

Hi all,

We are working with the Nextera Tagmentation Library Kit on 3 kb PCR products, to eventually pool libraries of 96 PCR reactions for a MiSeq run.

Tagmentation is done by plainly adding the enzyme + buffer to a dilution of the PCR reaction (no purification).

This generally works very well. However, for my last two library preps, my bioanalyzer traces ended up with a large peak at 3 kb (likely the original input), and nearly nothing in the 300-450 bp range.

This makes me believe that tagmentation was not effective.

Two potential culprits:

1) New PCR buffer (NEB Phusion HF Buffer) that includes detergent. Might not have been diluted enough and could have inhibited the transposon (final dilution of PCR buffer during tagmentation step was 1:25).


2) Loss of transposon activity. Kit technically expired six months ago; and it was accidentally thawed for several hours a few months ago. But we used it successfully after this accidental thaw.


I was thinking about an easy way to confirm that transposon activity is still there (without having immediate access to a bioanalyzer):

Purify PCR product > Tagmentation step according to Nextera protocol > Check whether the PCR band (ca. 3 kb) is gone (i.e., only smear or nothing detectable left).

Would that make sense to identify a loss in enzyme activity? Or would we not expect full tagmentation (i.e., complete loss of the 3 kb bnad) even for "fresh" enzyme.


Thanks!
sobbewoscht is offline   Reply With Quote
Old 05-25-2019, 05:48 PM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,223
Default

I would suggest to do a reaction according to standard protocol from start to end and check the resulting library. Tagmented DNA will not migrate according to size becuase:
1- transposon will be engaged with DNA and slow its migration speed
2- presence of salts in the buffer
nucacidhunter 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 04:39 PM.


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