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Old 03-28-2014, 09:16 AM   #1
PrawnFryan
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Location: Ireland

Join Date: Mar 2014
Posts: 1
Default Accidentally eluted with x100 Tris

Hi All,

I have just realised that at the end of the Ampure clean-up, I eluted with x100 Tris buffer by accident. Is this the source of my problems? And is there any way to solve this??
I have 120 human feacal samples which I am preparing for MiSeq at the moment. I am following the Illumina protocol and have the first PCR and clean-up completed (with very good PCR products).

I attempted to run the first plate with the Nextera library prep primers yesterday and when I ran the gel all I got back was a very faint band around where the first (16s amplicon) PCR product was (550bp) and no bands at all where they should be (630bp).

After rerunning 4 of my samples on a different PCR block and getting the same result, I gave a couple samples to my colleague who was doing a Nextera library prep and offered to try them. She got the same result and her prep didnt work.

Thank you!
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Old 03-31-2014, 12:00 PM   #2
microgirl123
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Location: New England

Join Date: Jun 2012
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Are you running the Illumina metagenomics protocol, and do you mean you eluted your first PCR rxn in 100x Tris? That's a lot of Tris to be putting into your second PCR rxn!

If this is the case, I'd try "recleaning" a couple samples from your first reaction and eluting into the proper Tris concentration. Then try the second PCR again.
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