illumina alternative v1.5 protocol for small rna seq vs. the standard protocol

ik76 · 02-25-2010, 08:42 AM

Hi All,
I was just wandering whether anybody had successfully used the illumina alternative v1.5 small RNA sample prep protocol . We tried but the only thing we could detect was adapter contamination.
If anybody has used it successfully can they comment on the 3' adapter ligation protocol ( the one using T4 RNA ligase 2 truncated). I was wandering why the portocol uses such a high Mg concentration ( 10mM I believe) and does not use PEG.

Bioo Scientific · 03-25-2010, 03:24 PM

Where did you get your adapter? What concentration of adapter did you use and how many units of ligase did you use? Adding too much adapter may lead to primer dimers and other artificial PCR products.

We have used the v1.5 sample prep protocol using our own enzyme, AIR Ligase, a truncated T4 RNA Ligase 2 successfully with our adenylated adapters. We would be happy to send you our recommended protocol and a sample of T4 RNA Ligase 2 (truncated).

Dawn Obermoeller
Bioo Scientific

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02-25-2010, 08:42 AM	#1
ik76 Junior Member Location: London, UK Join Date: Jan 2010 Posts: 9	illumina alternative v1.5 protocol for small rna seq vs. the standard protocol Hi All, I was just wandering whether anybody had successfully used the illumina alternative v1.5 small RNA sample prep protocol . We tried but the only thing we could detect was adapter contamination. If anybody has used it successfully can they comment on the 3' adapter ligation protocol ( the one using T4 RNA ligase 2 truncated). I was wandering why the portocol uses such a high Mg concentration ( 10mM I believe) and does not use PEG.

03-25-2010, 03:24 PM	#2
Bioo Scientific Registered Vendor Location: Austin, Tx Join Date: Oct 2009 Posts: 99	Troubleshooting v1.5 sample prep protocol Where did you get your adapter? What concentration of adapter did you use and how many units of ligase did you use? Adding too much adapter may lead to primer dimers and other artificial PCR products. We have used the v1.5 sample prep protocol using our own enzyme, AIR Ligase, a truncated T4 RNA Ligase 2 successfully with our adenylated adapters. We would be happy to send you our recommended protocol and a sample of T4 RNA Ligase 2 (truncated). Dawn Obermoeller Bioo Scientific Last edited by Bioo Scientific; 09-15-2015 at 11:49 AM.