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Old 10-12-2015, 04:22 AM   #163
jwfoley
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Location: Stanford

Join Date: Jun 2009
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Quote:
Originally Posted by JJMS View Post
Our profiles with high input and SMARTscribe indeed match ssII nearly to a 100%. But with single cells SMARTscribe deviates a little. I have to leave out Mg because otherwise a smear around 500 bp's becomes visible.
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/ÁL vs. SuperScript II's 200 U/ÁL.
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