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Old 12-18-2015, 11:15 AM   #4
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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.

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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