Inconsistent Yield With Nextera XT

BigAlPal · 07-18-2014, 01:39 AM

Hey all,

I've been using the Nextera XT kit without bead normalization and have been getting inconsistent yields after the PCR step. There doesn't seem to be any rhyme or reason to it. Right now, I'm trying to get some decent libraries with plasmids. Did 5 preps and only two of them were above 2nM. I've had the same issue with bacterial genomes and commercially prepared human genome. We've had limited success increasing input to 6ng and using 90ul Ampure beads but that too is inconsistent.

Has anybody had the same problem and if so, what have you done to fix it? I've been advised by another lab to switch to the regular Nextera kit but money is an issue with our lab. If we can make the cheaper Nextera XT kit work consistently, it would be better. Any advice would be appreciated!

BigAlPal

nucacidhunter · 07-18-2014, 02:38 AM

In my experience two important factors affecting success and consistency using Nextera based kits are DNA quantity and quality. Cleaning up DNA with columns or bead followed by fluorometric quantitation in two steps ensures yield of 1 ng/ul or higher in PCR which is sufficient for multiplex sequencing. Some DNA samples will do fine without clean up, but addition of this step reduces likelihood of failure.

BigAlPal · 07-18-2014, 11:50 AM

Thanks for your reply nucacidhunter. I was able to get it to work using 6ng input and increased the number of PCR cycles to 15. Got excellent concentrations and beautiful peaks on the tapestation. Not sure why I have to use so much more than the protocol asks for. Maybe because I'm working with plasmids, which are small? Had the same issue with larger, bacterial genomes too but nevertheless, that's what I'll be doing from now on.

snetmcom · 07-18-2014, 05:58 PM

How are you quantifying your starting DNA? Nextera is very sensitive to starting amount.

BigAlPal · 07-19-2014, 01:39 AM

Quote:

Originally Posted by snetmcom

How are you quantifying your starting DNA? Nextera is very sensitive to starting amount.

Qbit ds HS assay.

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07-18-2014, 01:39 AM	#1
BigAlPal Junior Member Location: Southeast U.S. Join Date: Aug 2013 Posts: 6	Inconsistent Yield With Nextera XT Hey all, I've been using the Nextera XT kit without bead normalization and have been getting inconsistent yields after the PCR step. There doesn't seem to be any rhyme or reason to it. Right now, I'm trying to get some decent libraries with plasmids. Did 5 preps and only two of them were above 2nM. I've had the same issue with bacterial genomes and commercially prepared human genome. We've had limited success increasing input to 6ng and using 90ul Ampure beads but that too is inconsistent. Has anybody had the same problem and if so, what have you done to fix it? I've been advised by another lab to switch to the regular Nextera kit but money is an issue with our lab. If we can make the cheaper Nextera XT kit work consistently, it would be better. Any advice would be appreciated! BigAlPal

07-18-2014, 02:38 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	In my experience two important factors affecting success and consistency using Nextera based kits are DNA quantity and quality. Cleaning up DNA with columns or bead followed by fluorometric quantitation in two steps ensures yield of 1 ng/ul or higher in PCR which is sufficient for multiplex sequencing. Some DNA samples will do fine without clean up, but addition of this step reduces likelihood of failure.

07-18-2014, 11:50 AM	#3
BigAlPal Junior Member Location: Southeast U.S. Join Date: Aug 2013 Posts: 6	Thanks for your reply nucacidhunter. I was able to get it to work using 6ng input and increased the number of PCR cycles to 15. Got excellent concentrations and beautiful peaks on the tapestation. Not sure why I have to use so much more than the protocol asks for. Maybe because I'm working with plasmids, which are small? Had the same issue with larger, bacterial genomes too but nevertheless, that's what I'll be doing from now on.

07-18-2014, 05:58 PM	#4
snetmcom Senior Member Location: USA Join Date: Oct 2008 Posts: 158	How are you quantifying your starting DNA? Nextera is very sensitive to starting amount.