Blue:RNA-Seq Experiments:11182013: Difference between revisions

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>B1lake
>B1lake
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#TSO (no PAP)
#TSO (no PAP)
#TSO + PAP
#TSO + Affy PAP
#TSO + PAP + 3'dATP
#TSO + Affy PAP + 3'dATP
#TSO + PAP + dATP
#TSO + Affy PAP + dATP
#TSO + PAP + dNTP (similar conc as RT reaction)
#TSO + Affy PAP + dNTP (similar conc as RT reaction)
#TSO + RNaseIII + PAP
#TSO + RNaseIII + Affy PAP
#TSO + RNaseIII + PAP (no Rnase Inhibitor)
#TSO + RNaseIII + Affy PAP (no Rnase Inhibitor)




Line 40: Line 40:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|10x RNaseIII buffer or 5x PAP buffer||||||||||0.2ul or 0.4ul
|10x RNaseIII buffer||||||||||0.2ul  
|-
|-
|RNase III||||||||||0.1ul
|RNase III or dH2O||||||||||0.1ul
|-
|-
|dH20||||||||||0.8ul or 0.6ul
|dH20||||||||||0.7ul
|-
|-
|Total||||||||||2ul
|Total||||||||||2ul
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|TSO||||||||||2ul
|TSO||||||||||2ul
|-
|-
|5x RT buffer or 5x Affy PAP Buffer||||||||||0.5ul or 0.4ul
|5x RT buffer||||||||||0.5ul
|-
|-
|1mM ATP (or mix)||||||||||0.5ul   
|1mM ATP (or mix)||||||||||0.5ul   
|-
|-
|2U/ul Ambion PAP or 600U/ul Affymetrix PAP||||||||||0.2ul   
|60U/ul Affymetrix PAP[1:10]||||||||||0.2ul   
|-
|-
|40U/ul Rnase Inhibitor||||||||||0.2ul
|40U/ul Rnase Inhibitor||||||||||0.2ul
|-
|-
|dH2O||||||||||0.6ul or 0.7ul
|dH2O||||||||||0.2ul
|-
|-
|dNTP mix||||||||||0.4ul
|dNTP mix or dH2O||||||||||0.4ul
|-
|-
|Total||||||||||4ul  
|Total||||||||||4ul  

Revision as of 22:40, 21 November 2013

Purpose

  • Test Affymetrix PAP on TSO poly(A) tailing using similar protocol as 11-13-2013
  • Test ability of [dATP] in the RT mix for inhibition of PAP activity
  • Test whether RNaseIII retains activity on Poly(A) tailed RNA


RNAseIII-Fragmented Library Preparation

Conditions
  1. TSO (no PAP)
  2. TSO + Affy PAP
  3. TSO + Affy PAP + 3'dATP
  4. TSO + Affy PAP + dATP
  5. TSO + Affy PAP + dNTP (similar conc as RT reaction)
  6. TSO + RNaseIII + Affy PAP
  7. TSO + RNaseIII + Affy PAP (no Rnase Inhibitor)


RNA
Volume
1uM TSO.r04 1ul
Total 1ul


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


Volume
10x RNaseIII buffer 0.2ul
RNase III or dH2O 0.1ul
dH20 0.7ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
TSO 2ul
5x RT buffer 0.5ul
1mM ATP (or mix) 0.5ul
60U/ul Affymetrix PAP[1:10] 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.2ul
dNTP mix or dH2O 0.4ul
Total 4ul


Incubate @ 37C 10 min