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

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[[File:10232013_gel2.jpg]]
[[File:10232013_gel2.jpg]]
== Ambion PAP Tests ==
*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
**Ambion PAP 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
:'''totoRNAseq Reaction Buffer'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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
|-
|}
:'''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 01:32, 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


Protocol

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


Ambion PAP Tests

  • 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
    • Ambion PAP 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



totoRNAseq Reaction Buffer


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



Ambion Reaction Buffer


Volume
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