PCR amplicon normalization

_anli · 05-13-2019, 06:29 AM

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!

r.rosati · 05-13-2019, 02:47 PM

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.

_anli · 05-13-2019, 11:54 PM

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?

r.rosati · 05-14-2019, 06:21 PM

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).

luc · 05-14-2019, 08:29 PM

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.

ikripp · 05-15-2019, 04:12 PM

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.

ATϟGC · 05-24-2019, 09:32 AM

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.

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05-13-2019, 06:29 AM	#1
_anli Junior Member Location: Sweden Join Date: Aug 2018 Posts: 3	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!

05-13-2019, 02:47 PM	#2
r.rosati Member Location: Brazil Join Date: Aug 2015 Posts: 94	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.

05-13-2019, 11:54 PM	#3
_anli Junior Member Location: Sweden Join Date: Aug 2018 Posts: 3	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?

05-14-2019, 06:21 PM	#4
r.rosati Member Location: Brazil Join Date: Aug 2015 Posts: 94	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).

05-14-2019, 08:29 PM	#5
luc Senior Member Location: US Join Date: Dec 2010 Posts: 453	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.

05-15-2019, 04:12 PM	#6
ikripp Member Location: QLD, Aus Join Date: Jan 2018 Posts: 19	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.

05-24-2019, 09:32 AM	#7
ATϟGC Member Location: Canada Join Date: Jun 2013 Posts: 56	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.