Blue:RNA-Seq Experiments:10232013

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Revision as of 21:08, 24 October 2013 by >B1lake (→‎Adaptation to totoRNAseq)
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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


File:10232013 gel3.jpg

Adaptation to totoRNAseq

  • Test:
    • Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
      • Commercial mix Ambion (see above)
        • 10mM ATP
        • 1mM ATP
        • 1mM ATP/0.1mM 3' d-ATP
        • 1mM ATP/0.5mM 3' d-ATP
      • totoRNAseq mix Ambion (below)
        • 10mM ATP
        • 1mM ATP
        • 1mM ATP/0.1mM 3' d-ATP
        • 1mM ATP/0.5mM 3' d-ATP
      • totoRNAseq mix Enzymatics (below)
        • 10mM ATP
        • 1mM ATP
        • 1mM ATP/0.1mM 3' d-ATP
        • 1mM ATP/0.5mM 3' d-ATP


totoRNAseq Reaction Buffer
Volume
1uM TSO.r04 1ul
dH20 6.4ul
5x RT buffer 1.25ul
10x RnaseIII buffer 0.5ul
1mM ATP or 10mM ATP 0.625ul
0.5U PAP 0.25ul