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Old 05-22-2017, 06:19 AM   #13
Simone78
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Location: Bonn (Germany)

Join Date: Oct 2010
Posts: 189
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Quote:
Originally Posted by Arthur45 View Post
Dear Simone and other Nextera users,

I was wondering how to go about the Tagmented DNA Clean-up in the Nextera protocol. It uses a Zymo spin column, which I guess will not be possible for a smaller volume (e.g. 2-10 ul). What do you do at this stage? Do you clean with beads? Skip the whole clean-up? Dilute the Tagmented DNA in buffer to make up for the original 50ul volume needed?

Thanks,

Arthur


Would it b

Hi Arthur,
no need to clean up the tagmented DNA before the final PCR. I do the tagmentation reaction in 2 ul, add 0.5 ul of NT buffer (or 0.2% SDS) and simply go on with the PCR mix (2.5 ul). 0.2% SDS will remove the Tn5 from the DNA and, after adding the PCR mix, will be diluted down and won´t interfere with the final PCR.
Only after PCR you will do a clean up and, if you want to multiplex tens or hundreds of cells, I would suggest to pool all of them and then do the cleanup, not the other way around.
Best,
Simone
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