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-   -   Neutralize Tagment Buffer (NT) (http://seqanswers.com/forums/showthread.php?t=50392)

michael_liu 02-17-2015 07:12 PM

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!

nucacidhunter 02-17-2015 08:33 PM

I wonder what evidence you have for your conclusion. It is an unlikely event.

Simone78 02-18-2015 06:34 AM

Quote:

Originally Posted by michael_liu (Post 160437)
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

cmbetts 12-18-2015 11:15 AM

Quote:

Originally Posted by Simone78 (Post 160500)
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.

cmbetts 12-18-2015 12:04 PM

Nevermind. Found the official stuff.

Adz 10-18-2019 01:46 AM

Yes
 
Quote:

Originally Posted by cmbetts (Post 186605)
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.

I've tried and it works perfectly.


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