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Old 06-02-2015, 04:15 PM   #1
NYGen
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Default After 1st-strand cDNA synthesis for RNAseq, is it safe to wait a day for 2nd?

Greetings All,

It's my first time doing library prep for RNAseq on a eukaryotic organism, and unfortunately I failed to order 2nd strand cDNA synthesis mix before the end of business today, so there's no way that I'll get the mix tomorrow. However, I do have the materials needed 1st strand cDNA synthesis.

I fully realize that 24 hours is not a long time to wait to get this library prepared, but I'm actually in a time crunch to get this sent to the sequencing core and I want to optimize for time. On that basis, is it safe to synthesize the 1st cDNA strand, and then leave the samples overnight in cold storage while I wait for the 2nd cDNA strand synthesis mix to arrive the following morning?

Of course, prior to posting a new question I searched for an answer to my question. I didn't find anything here or elsewhere that directly addresses this issue. The closest I found was this protocol which says at the end of the section regarding 1st strand synth: "After incubation at 4°C, centrifuge the tube briefly (~5 seconds) to collect the reaction at the bottom of the tube and immediately proceed to Step 3: Second-Strand cDNA Synthesis." which is clearly mix-specific, as an alternative protocol from the same document only states the need to incubate the tubes at 42C for an hour prior to proceeding to the synth of 2nd strand.

In short, is it safe to wait? Ultimately it's just cDNA and mRNA, right? Happy to receive any insights- thanks for your help!
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Old 06-03-2015, 12:15 AM   #2
Simone78
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Itīs perfectly fine, donīt worry! If you make cDNA using a strand-switch oligo (Smarter, Smart-seq2, STRT methods) you might notice a lower yield if you leave the 1st strand reaction in the fridge for a week or more before doing the PCR (donīt know the reason but this is what we observed), but nothing bad happens if you leave it overnight. Even if you use another system (DNA Pol I, RNAse H) there shouldnīt be any problems.
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Old 06-03-2015, 10:57 AM   #3
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Perfect, thanks very much for the explanation. Cheers!
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