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-   -   illumina alternative v1.5 protocol for small rna seq vs. the standard protocol (http://seqanswers.com/forums/showthread.php?t=4172)

ik76 02-25-2010 07:42 AM

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.

Bioo Scientific 03-25-2010 02:24 PM

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


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