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Old 04-09-2012, 05:22 AM   #6
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Location: france

Join Date: Mar 2012
Posts: 7

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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