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-   -   Questions about Nextera kit/tagmentation (http://seqanswers.com/forums/showthread.php?t=72196)

kobeho24 10-26-2016 02:00 AM

Questions about Nextera kit/tagmentation
 
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Hi folks,
Just wonder if anyone has an experience of using the Nextera kit or home-made tagmentation assay based on the Genome Research paper. http://genome.cshlp.org/content/earl....full.pdf+html
We are not able to get libraries as expected with home made version, in particular the fragment size is hard to manipulate according to the protocol attached in the paper. (adjust input amount, modify reaction buffer composition, etc. none of them works) :(
But anyway, we can get amplicon after PCR with KAPA HiFi kit.
However, when we start trying to do some ATAC-seq with home-made Tn5, any purification step between tagmentation and PCR turned out to have undetectable amplicon after PCR. Whereas simply adding 0.1%-0.2% SDS to the tagmented product prior to PCR can give us a whole bunch of DNA after all.
If anyone can give me some idea, that would be much appreciated!!! :)

Gary

pmiguel 10-26-2016 11:33 AM

I've read that the Tn5 transposase stays bound to both ends of the DNA that it has cleaved. Perhaps the purification step removes both the protein (transposase) and the DNA it is attached to. Whereas treatment with SDS could denature the transposase, allowing the DNA to be recovered?

--
Phillip

kobeho24 10-26-2016 08:09 PM

Quote:

Originally Posted by pmiguel (Post 200364)
I've read that the Tn5 transposase stays bound to both ends of the DNA that it has cleaved. Perhaps the purification step removes both the protein (transposase) and the DNA it is attached to. Whereas treatment with SDS could denature the transposase, allowing the DNA to be recovered?

--
Phillip

Hi Phillip,
Thanks for your reply. It may explain part of my confusion. But how come I cannot get a detectable mount of DNA with the condition of using SDS prior to beads purification? That doesn't make sense. :(
And the most important thing is that there isn't any SDS treatment but purification step in Nextera DNA prep kit, and it must work, otherwise illumina can't make it on the market.

Gary

pmiguel 10-27-2016 05:21 AM

I don't know. Does SDS prevent DNA from binding to Ampure beads, perhaps?

Nextera may have additives in its buffers, or even a mutated Tn5 transposase that performs differently.

--
Phillip

kobeho24 10-27-2016 08:01 PM

Quote:

Originally Posted by pmiguel (Post 200405)
I don't know. Does SDS prevent DNA from binding to Ampure beads, perhaps?

Nextera may have additives in its buffers, or even a mutated Tn5 transposase that performs differently.

--
Phillip

I have no idea about that either. But I already super diluted the reaction containing limited amount of SDS prior to beads purification~ I suppose it should be the buffer issue, somehow illumina just has tricks~

Gary


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