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Old 02-17-2015, 07:12 PM   #1
michael_liu
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Default Neutralize Tagment Buffer (NT)

Hi Everyone, Does anyone have a recipe for the Neutralize Tagment Buffer (NT) that comes with Nextera XT DNA Sample Preparation Kit? Thanks heaps!

Last edited by michael_liu; 02-17-2015 at 08:40 PM.
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Old 02-17-2015, 08:33 PM   #2
nucacidhunter
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I wonder what evidence you have for your conclusion. It is an unlikely event.
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Old 02-18-2015, 06:34 AM   #3
Simone78
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Quote:
Originally Posted by michael_liu View Post
Hi Everyone, Does anyone have a recipe for the Neutralize Tagment Buffer (NT) that comes with Nextera XT DNA Sample Preparation Kit? Thanks heaps!
The NT buffer (MOST LIKELY) is 0.2% SDS, as we also described in our recent paper for home-made Tn5 (Picelli et al., Genome Res 2014).
In principle, 0.1%-0.2% SDS will work. Increasing the conc to 0.3% SDS led to a failure of the follwoing PCR reaction (for us, at least).

We add 5 ul of 0.2% SDS to the 20 ul Tn5 reaction, incubate 5 min at RT (probably not necessary) and then do the PCR in 50 ul (same as the Nextera XT kit), which means you have 0.02% SDS in the final PCR reaction. The rationale behind that is to have a detergent that strips the Tn5 off the the DNA but gets then diluted and doesn´t kill the Polymerase.
I guess that "extensive" heating at 72 degrees (10 min? 15? longer?) could also work. But SDS is the quick and safe alternative.

/Simone
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Old 12-18-2015, 11:15 AM   #4
cmbetts
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Quote:
Originally Posted by Simone78 View Post
The NT buffer (MOST LIKELY) is 0.2% SDS, as we also described in our recent paper for home-made Tn5 (Picelli et al., Genome Res 2014).
In principle, 0.1%-0.2% SDS will work. Increasing the conc to 0.3% SDS led to a failure of the follwoing PCR reaction (for us, at least).

We add 5 ul of 0.2% SDS to the 20 ul Tn5 reaction, incubate 5 min at RT (probably not necessary) and then do the PCR in 50 ul (same as the Nextera XT kit), which means you have 0.02% SDS in the final PCR reaction. The rationale behind that is to have a detergent that strips the Tn5 off the the DNA but gets then diluted and doesn´t kill the Polymerase.
I guess that "extensive" heating at 72 degrees (10 min? 15? longer?) could also work. But SDS is the quick and safe alternative.

/Simone
Has anyone tested this with the official Nextera XT kit? The RT storage components of my Nextera kit wandered off while I was away from the bench for a while... I'm going to dig around the lab some more, and try out 0.2% SDS if I'm unsuccessful. I've got enough kit and sample to risk it.
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Old 12-18-2015, 12:04 PM   #5
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Nevermind. Found the official stuff.
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