Blue:RNA-Seq Experiments:11062013: Difference between revisions
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>B1lake (Created page with "== Purpose == *Use bioanalyzer to test different fragmentation/poly(A) conditions - continuation on 11052013 == RNAseIII-Fragmented Library Preparation == :'''Conditions...") |
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:::''Incubate @ 37C 10 min - 30 min'' | :::''Incubate @ 37C 10 min - 30 min'' | ||
:::''Column Purify Samples'' | :::''Column Purify Samples'' | ||
Bioanalyzer: |
Revision as of 18:24, 8 November 2013
Purpose
- Use bioanalyzer to test different fragmentation/poly(A) conditions - continuation on 11052013
RNAseIII-Fragmented Library Preparation
- 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: