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Old 04-06-2012, 12:45 PM   #5
thedavid
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Location: MD

Join Date: Jul 2011
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Has anyone not in Hinrich Gronemeyer's lab gotten this process to work? I've been trying for well over a month to get it working. After a tremendous amount of troubleshooting I've made a few discoveries.

First, an aside: you can't buy the SAP they recommend (not sold anymore). The only one I can find still for sale is from USB. I assume a recombinant SAP cannot be used because they seem far pickier about the buffers used.

I have great difficulty getting the T-tailing and T7-BpmI-oligoA(15) addition to work. I discovered this week I get far better efficiency when I skip the SAP reaction.

Here's an example:


Lane 2 and Lane 3 are identical, except that Lane 2 had the SAP reaction done. And yes, I did heat inactivate at 70 C for 10 min.

Still, as you can see from the doublet in Lane 3, I'm not getting full linker addition. It looks about 50% efficient. The authors show a near 100% efficiency in a supplemental figure, so I obviously have some improving to do.

I took two of the reactions (set up like Lane 3), one with 50 PICOgrams and the other with 50 NANOgrams of the PCR product and proceeded forward with the in vitro transcription.

From the 50 ng input, I got a pretty good RNA yield (assayed by NanoDrop) - 580 ng/ul in 30 ul elution. For the 50 pg input I got a terrible to non-existant yield - 3 ng/ul in 30 ul elution. I haven't had the opportunity to confirm these yields on a BioAnalyzer. I should be able to do that next week. From experience, I am quite confident that 3ng/ul is really nothing.

I'm assuming my yield from the 50 pg input is so poor because of the inefficient T7 oligo addition.

Sorry for all of the rambling. Does anyone have any tips for me?

I'm using aliquoted T-mix, dNTPs, and T7-BpmI-oligoA(15), to reduce freeze/thaw degradation. The T7-BpmI-oligoA(15) was synthesized by IDT DNA and PAGE purified. All of the reagents (except the SAP, are the same brand/type as recommended in the protocol). I'm following the Nature Methods papers exactly, except that I'm adding 2 ul of 2.5 mM CoCl2 (the protocol calls for adding 1 ul of 5 mM CoCl2).
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