SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Preparation for amplicon sequencing without PCR GMDickson Illumina/Solexa 13 07-28-2017 02:30 AM
Amplicon normalization RCJK Sample Prep / Library Generation 3 06-20-2013 05:55 AM
coligation of PCR amplicon amhro Sample Prep / Library Generation 3 09-26-2012 05:12 AM
Normalization of 16S PCR products cmazzoni Metagenomics 0 07-21-2011 05:17 AM
PCR product normalization Palecomic Sample Prep / Library Generation 0 01-20-2011 01:50 AM

Reply
 
Thread Tools
Old 05-13-2019, 05:29 AM   #1
_anli
Junior Member
 
Location: Sweden

Join Date: Aug 2018
Posts: 3
Default PCR amplicon normalization

Hi all.

I am looking for robust ways of normalizing concentrations of PCR amplicons.
I've previosuly done this by concentration measurement using either a nanodrop/qubit or from band intensity from gel electrophoresis.

I'm now looking into using magnetig beads (SPRI) insetad. The standard beads (like AMPure XP) are in excess when doing the regular PCR clean-up protocols, but thought that perhaps one could dilute them somehow in order to get a pre-determined output concentrations. From this one could also use home-made beads in order to reduce cost.

Does anybody have any experience in doing these sort of things?

Thanks!
_anli is offline   Reply With Quote
Old 05-13-2019, 01:47 PM   #2
r.rosati
Member
 
Location: Brazil

Join Date: Aug 2015
Posts: 89
Default

This is basically the idea behind ThermoFisher's "Equaliser" NGS kit. In our hands it didn't work particularly well, though. PCR is really more accurate.
r.rosati is offline   Reply With Quote
Old 05-13-2019, 10:54 PM   #3
_anli
Junior Member
 
Location: Sweden

Join Date: Aug 2018
Posts: 3
Default

Thanks for the feedback! I've seen that there are several kits doing this sorta thing out there and I've been curious about peoples results.

What did you mean by PCR being more accurate?
_anli is offline   Reply With Quote
Old 05-14-2019, 05:21 PM   #4
r.rosati
Member
 
Location: Brazil

Join Date: Aug 2015
Posts: 89
Default

I'm sorry, did I misunderstand you? I thought you were considering libraries, and then what I meant is that qPCR with adaptor-specific primers is a better option than equalizing with SPRI beads. The concentration you get from qPCR is fairly accurate, and you can mix your amplicons in equimolar amount with good success. With SPRI beads we noticed that the amounts often weren't much equimolar.
If you mean regular PCR amplicons, I never trusted spectrophotometry below 20 ng/ul. I also used to quantify by gel, but currently I use spectrofluorimetry (a Qubit specifically, it works fine but I'm sure other brands work just as well).
r.rosati is offline   Reply With Quote
Old 05-14-2019, 07:29 PM   #5
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 407
Default

I do not believe that the bead surface is strongly limiting the DNA amounts that can be precipitated onto it - at least not when using standard Ampure (PEG/NaCl) buffer.
It is worth a try.
luc is offline   Reply With Quote
Old 05-15-2019, 03:12 PM   #6
ikripp
Member
 
Location: QLD, Aus

Join Date: Jan 2018
Posts: 19
Default

Our workflow uses the Agilent tapestation to quantify the PCR products which are then normalised from this and pooled as required. There are some limitations to the accuracy of this method but provided that all samples are checked on the same machine/same tape type etc. it works quite well.
ikripp is offline   Reply With Quote
Old 05-24-2019, 08:32 AM   #7
ATϟGC
Member
 
Location: Canada

Join Date: Jun 2013
Posts: 48
Default

I tried to use limiting amount of SPRI beads (Sera-Mag speedbeads and Commercial preparations diluted in their own buffer) for the normalization of amplicons prior to pooling without any success. I always found that the normalization was poor and that the amount of DNA eluted at the end was somewhat proportional to the starting concentration of the DNA. I gave up on it after a few days. Please post your protocol if you find something that works.
ATϟGC 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 02:33 PM.


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