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Old 05-23-2017, 08:25 AM   #14
Arthur45
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Location: London

Join Date: May 2017
Posts: 7
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Quote:
Originally Posted by Simone78 View Post
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
Hi Simone,

thank you for the message - this is so much easier than expected!

You mention after the PCR I should do a bead clean-up. The original protocol mentions to add twice the beads to the PCR product :

4 Add 30 μl AMPure XP beads to NAP2.
5 Add 30 μl AMPure XP beads to NAP2.
To me, this ratio of 20:30+30 looks like a mistake. Say I have 11ul of PCR products, would I add twice 16.5ul?

I hope someone can help me out there.

Cheers,
Arthur
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