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Old 03-31-2018, 08:24 AM   #1
achamess
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Default Would Tn5 (Nextera) work on a ~100 bp fragment?

I'm using Nextera to tagment full length cDNA from a heterogeneous mixture. Within this mix, there should also be a small dsDNA fragment that is 113 bp long. I do not want the Tn5 to do anything to this small fragment. I don't need it tagmented. My plan is to separate this 113 bp fragment from the longer tagmented cDNAs by Ampure XP, and take the supernatant (containing the small fragment).

The Nextera manual says Tn5 doesn't work on amplicons < 300 bp, but I'm not sure about that. does anyone have any thoughts on this?

Thanks.
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Old 03-31-2018, 10:22 AM   #2
luc
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It is unlikely that the Tn5 will cut/tagment your 113 bp fragment twice - this would be required for sequencing.
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Old 03-31-2018, 12:18 PM   #3
achamess
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Quote:
Originally Posted by luc View Post
It is unlikely that the Tn5 will cut/tagment your 113 bp fragment twice - this would be required for sequencing.
Thanks!
How about not at all? I suppose there is no way to know but try. But I'm actually trying to recover this fragment untouched by Tn5. That might not be possible. I may need to recover it before Tn5. But we'll see...
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Old 03-31-2018, 07:54 PM   #4
nucacidhunter
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Nextera will cut smaller DNA (I think it is above 30 bp long) fragments as well as large ones but the chance of short fragments being cut at both ends and to be in the included size range in final library is lower than large fragments.

Your plan of separating short fragments from the cDNA by AMPure before tagmentation is fine. You still will loose some short fragments by recovering from supernatant that can be minimized by increasing final bead to cDNA solution ratio.
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Old 04-03-2018, 12:56 AM   #5
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Just to make sure you are aware:
The Tn5 does not only fragment your DNA, it also adds primer binding sites that are essential for the downstream Nextera workflow. In your post, it sounds as if you want to "rescue" the small fragments from fragmentation and re-introduce them into the Nextera Workflow after the Transposase step. This will not work because the index PCR will not amplify these "untouched" fragments.
The only way for you to sequence these small fragments is the classical "End Repair / A-Tailing / Adapter-Ligation" workflow.
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Old 04-30-2018, 11:49 PM   #6
Simone78
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Quote:
Originally Posted by Genetic Librarian View Post
Just to make sure you are aware:
The Tn5 does not only fragment your DNA, it also adds primer binding sites that are essential for the downstream Nextera workflow. In your post, it sounds as if you want to "rescue" the small fragments from fragmentation and re-introduce them into the Nextera Workflow after the Transposase step. This will not work because the index PCR will not amplify these "untouched" fragments.
The only way for you to sequence these small fragments is the classical "End Repair / A-Tailing / Adapter-Ligation" workflow.
I also see another problem here. A 113-bp fragments is very close to the cutoff of the beads. I am not sure youŽll be able to fish it out in an efficient way. Moreover, trying to avoid the tagmentation of a 113-bp fragment might require some titration, as the Tn5 can cut fragments as short as 38 bp.
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