Blue:RNA-Seq Experiments:10232013: Difference between revisions

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[[File:10232013_gel2.jpg]]
[[File:10232013_gel2.jpg]]


== Ambion PAP Tests ==
== Poly A Limiting Tests ==


:'''3'dATP Blocking'''


*Test:
*Test:
**Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
**Ambion PAP (0.4U) with 3'dATP
***Commercial mix (see above)
***1mM ATP/0mM 3'ATP
***totoRNAseq mix with 1mM ATP
***1mM ATP/0.01mM 3'ATP
***totoRNAseq mix with 10mM ATP
***1mM ATP/0.05mM 3'ATP
***Commercial mix pre-heat inactivated
***1mM ATP/0.1mM 3'ATP
**Ambion PAP with 3'dATP
***1mM ATP/0.5mM 3'ATP
***0mM ATP/1mM 3'ATP
**Affymetrix PAP (60U)with 3'dATP
***1mM ATP/0mM 3'ATP
***1mM ATP/0mM 3'ATP
***1mM ATP/0.01mM 3'ATP
***1mM ATP/0.01mM 3'ATP
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== Adaptation to totoRNAseq ==


:'''totoRNAseq Reaction Buffer'''
:'''totoRNAseq Reaction Buffer'''
*Test:
**Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
***Commercial mix (see above)
***totoRNAseq mix with 1mM ATP
***totoRNAseq mix with 10mM ATP
***Commercial mix pre-heat inactivated




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|-
|-
|0.5U PAP||||||||||1ul
|0.5U PAP||||||||||1ul
|-
|}
:'''Ambion Reaction Buffer'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|1uM TSO.r04||||||||||1ul
|-
|dH20||||||||||4.6ul
|-
|5x E-PAP buffer||||||||||2ul
|-
|25mM MnCl2||||||||||1ul
|-
|1mM ATP +/- 3'dATP||||||||||1ul
|-
|1U/ul PAP [1:5 dil]||||||||||1ul
|-
|-
|}
|}

Revision as of 19:30, 24 October 2013

Purpose

  • Test PAP Activity from different companies:
    • Ambion
    • Enzymatics (used up to this point)
    • Affymetrix
  • Test Blocking of PAP activity using Cordycepin-ATP


Commercial Protocols

Ambion 3' Tailing
Volume
1uM TSO.r04 1ul
dH20 4.6ul
5x E-PAP buffer 2ul
25mM MnCl2 1ul
10mM ATP 1ul
2U/ul PAP 0.4ul


Enzymatics 3' Tailing
Volume
1uM TSO.r04 1ul
dH20 6ul
10x PAP buffer 1ul
10mM ATP 1ul
0.5U/ul PAP (1:10 Dil) 1ul


Affymetrix 3' Tailing
Volume
1uM TSO.r04 1ul
dH20 5ul
5x PAP buffer 2ul
10mM ATP 1ul
60U/ul PAP (1:10 Dil) 1ul


Incubate @ 37C 0, 5, 10, 15, 20 min
Transfer to Ice


  • Run 5ul on urea gel:


File:10232013 gel1.jpg

File:10232013 gel2.jpg

Poly A Limiting Tests

3'dATP Blocking
  • Test:
    • Ambion PAP (0.4U) with 3'dATP
      • 1mM ATP/0mM 3'ATP
      • 1mM ATP/0.01mM 3'ATP
      • 1mM ATP/0.05mM 3'ATP
      • 1mM ATP/0.1mM 3'ATP
      • 1mM ATP/0.5mM 3'ATP
      • 0mM ATP/1mM 3'ATP
    • Affymetrix PAP (60U)with 3'dATP
      • 1mM ATP/0mM 3'ATP
      • 1mM ATP/0.01mM 3'ATP
      • 1mM ATP/0.05mM 3'ATP
      • 1mM ATP/0.1mM 3'ATP
      • 1mM ATP/0.5mM 3'ATP
      • 0mM ATP/1mM 3'ATP


Adaptation to totoRNAseq

totoRNAseq Reaction Buffer


  • Test:
    • Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
      • Commercial mix (see above)
      • totoRNAseq mix with 1mM ATP
      • totoRNAseq mix with 10mM ATP
      • Commercial mix pre-heat inactivated


Volume
1uM TSO.r04 1ul
dH20 4.6ul
5x RT buffer 1.25ul
10x RnaseIII buffer 0.5ul
1mM ATP or 10mM ATP 0.625ul
0.5U PAP 1ul