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...")
 
>B1lake
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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
  1. RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1mM ATP) 10min
  2. RNaseIII (5mM MgCl2)/PAP (2.5mM MnCl2 and 0.1mM ATP) 10min
  3. RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1:1 dATP) 10min
  4. RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2 and 1:1 dATP) 30min
  5. RNaseIII (10mM MnCl2)/PAP (5mM MnCl2 and 1:1 dATP) 10min
  6. RNaseIII (10mM MnCl2)/PAP (5mM MnCl2 and 1:1 dATP) 30min
  7. RNaseIII (20mM MnCl2)/PAP (10mM MnCl2 and 1:1 dATP) 10min
  8. 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: