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Old 10-12-2015, 08:12 AM   #164
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
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Quote:
Originally Posted by jwfoley View Post
SuperScript II's reaction buffer is 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2. ClonTech doesn't say what's in the SMARTScribe mix but according to the documentation from their "legacy" single-cell RNA-seq kit, the first-strand reaction buffer is (at 1X) 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2. If that's the same thing they provide when you buy SMARTScribe separately (though it might not be, since they also give you 20 mM DTT instead of 100 mM, as I hope you noticed), then of course you have to adjust the magnesium to compensate. Note that the SMARTScribe enzyme is also provided at 100 U/L vs. SuperScript II's 200 U/L.
thats true, thanks for the explanation. I should have said "repackaged Superscript...with a twist" (changed few details to justify the new packaging)
However, nowadays most or all of the RT protocols around are done with some kind of MMLV-based enzyme and wont differ so much from each other. My opinion, at least.
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