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Old 12-10-2015, 09:18 AM   #172
chaichontat
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Location: Bangkok

Join Date: Sep 2015
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Quote:
Originally Posted by Simone78 View Post
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
Hi Simone,

It turned out that my PCR primers were the problem. I was too fixated on the template switching process that I completely ignored the preamplification. Upon replacing the primers, everything works beautifully. I really thank you for this brilliant protocol!

Chaichontat
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