Blue:RNA-Seq Experiments:11062013
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Purpose[edit]
- Use bioanalyzer to test different fragmentation/poly(A) conditions - continuation on 11052013
RNAseIII-Fragmented Library Preparation[edit]
- Conditions
- RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1mM ATP) 10min
- RNaseIII (5mM MgCl2)/PAP (2.5mM MnCl2 and 0.1mM ATP) 10min
- RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1:1 dATP) 10min
- RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1:1 dATP) 30min
- RNaseIII (10mM MnCl2)/PAP (5mM MnCl2 and 1:1 dATP) 10min
- RNaseIII (10mM MnCl2)/PAP (5mM MnCl2 and 1:1 dATP) 30min
- RNaseIII (20mM MnCl2)/PAP (10mM MnCl2 and 1:1 dATP) 10min
- RNaseIII (20mM MnCl2)/PAP (10mM MnCl2 and 1:1 dATP) 30min
- RNA
Volume UHRR 10ng/ul 1ul 5x Ambion PAP buffer 0.08ul MnCl2 0.4ul RNase III 0.1ul dH20 0.42ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume fRNA 2ul 5x Ambion PAP Buffer 0.72ul ATP (or mix) 0.25ul 2U/ul PAP 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.63ul
- Incubate @ 37C 10 min - 30 min
- Column Purify Samples
Bioanalyzer:
File:2100 expert Eukaryote Total RNA Pico DE72903057 2013-11-08 08-37-03.pdf