Blue:RNA-Seq Experiments:11052013: Difference between revisions

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>B1lake
(Created page with "== Purpose == *Use bioanalyzer to test different fragmentation/poly(A) conditions == RNAseIII-Fragmented Library Preparation == :'''RNA''' ::{| {{table}} |- |||||||||||...")
 
>B1lake
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:::''Incubate @ 37C 5 min''
:::''Incubate @ 37C 5 min''


:::''Column Purify''




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|-
|-
|5x Ambion PAP Buffer||||||||||0.72ul  
|5x Ambion PAP Buffer||||||||||0.72ul  
|-
|25mM MnCl2||||||||||0.32ul
|-
|-
|ATP (or mix)||||||||||0.25ul   
|ATP (or mix)||||||||||0.25ul   
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|40U/ul Rnase Inhibitor||||||||||0.2ul
|40U/ul Rnase Inhibitor||||||||||0.2ul
|-
|-
|dH2O||||||||||0.31ul
|dH2O||||||||||0.63ul
|-
|-
|}
|}
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:::''Incubate @ 37C 10 min''
:::''Incubate @ 37C 10 min''
:::''Transfer to Ice''
:::''Column Purify''

Revision as of 21:20, 5 November 2013

Purpose

  • Use bioanalyzer to test different fragmentation/poly(A) conditions


RNAseIII-Fragmented Library Preparation

RNA
Volume
UHRR 10ng/ul 1ul
5x Ambion PAP buffer 0.08ul
dH20 0.09ul
Total 1.17ul


Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.17ul
25mM MnCl2 0.08ul
RNase III 0.1ul
dH20 up to 0.83ul
Total 2ul


Incubate @ 37C 5 min
Column Purify


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
Column Purify