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Old 04-09-2012, 07:45 AM   #7
thedavid
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Location: MD

Join Date: Jul 2011
Posts: 9
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1. Thanks, I'll give it a try.
2. Sorry, I wasn't clear - if the Klenow was added, then 10 mM dNTPs were added. What I meant was that 100 uM dNTPs were added instead of 100 uM T-mix. As a control, of course, since they wouldn't be very useful for adding the T7-BpmI-OligoA(15)
3. Perhaps.
4. That's true - I was using 50ng because it is difficult to visualize less on a gel.
5. We don't have a Qubit, but we do have BioAnalyzer Pico chips, which can measure in the hundreds of picograms of RNA.
6. Yes, I was using the SS III kit with the 10x buffer (hence I could use 26ul of RNA instead of 22ul), since that is what I had on hand. I'll switch to the kit with the 5x buffer in the future.

If you only expect 20ng of RNA total from 50pg of DNA, then I should expect to get only around 5-10ng of DNA in the end? Which would not be a challenging amount to build a library for next-gen sequencing. So I would need at around 200 pg of input into the system to comfortably generate enough material for next-gen sequencing?

Quote:
Originally Posted by linampli View Post
the detailed protocol is in nature protocols where the exact reaction volumes are given.
1. Unfortunately Promega seems to have discontinued SAP, however, the thermostable phosphatase seems to be a good alternative.
2. In your gel you mention that NO dNTP was added for the reactions in
lanes 1 2, 3. Is that right? If there is no dNTP added with the T7 oligo klenow would start chewing back the strand.
3. Perhaps there is something wrong with your SAP. The SAP step is expendable, ie. it is there only to improve the efficiency.
4. Try your reaction with 1 ng of DNA, 50 ng maybe too much substrate and that is why you see only 50% efficiency.
5. Do a Qubit HS for RNA quantitation, Qubit seems to be the most accurate and sensitive test for DNA/RNA. For 50 pg of substrate you should get around 10 to 20 ng of RNA (in 30 Ál), ie. 0.3 to 0.6 ng / Ál (nanodrop volume). this is far below the detection limit for nanodrop or for that matter bioanalyser.
6. so try Qubit, and elute in 20 Ál (imp for the next step)
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