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:
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
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
- 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