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Old 11-29-2015, 01:01 PM   #170
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 208
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Quote:
Originally Posted by chaichontat View Post
Hi Simone,

I used the Smart-seq2 protocol using the Superscript IV. For some reason, the reaction failed completely (no cDNA products were observed). When I did not add 1M betaine (Sigma) and 6mM MgCl2, the RT reaction went beautifully, but there's no template switching. The template was 10 ng of purified PBMCs RNA and the TSO was synthesized by Eurogentec. Could you provide me with the reaction conditions you used with SSRT IV?

Thanks!
Chaichontat
Hi,
I did exactly as described in the Nat Prot paper, just replacing SSRTII with SSRTIV and doing the RT for 15 min @ 50 degrees and inactivated the enzyme for 10 min @ 80 degrees.. I tested it only on tot RNA (10 pg) and I actually noticed that when I didnīt add betaine and MgCl2 I got worse results than when using them...the opposite of what you found. in my case the template switching went ok, at least based on the cDNA yield and size.
Best,
Simone
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