Blue:RNA-Seq Experiments:10232013: Difference between revisions
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[[File:10232013_gel4.jpg]] | [[File:10232013_gel4.jpg]] | ||
*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 -RT (below) | |||
****10mM ATP | |||
****1mM ATP | |||
****1mM ATP/0.1mM 3' d-ATP | |||
****1mM ATP/0.5mM 3' d-ATP | |||
***totoRNAseq mix -RT +NaCl (below) | |||
****10mM ATP | |||
****1mM ATP | |||
****1mM ATP/0.1mM 3' d-ATP | |||
****1mM ATP/0.5mM 3' d-ATP | |||
:'''totoRNAseq Reaction Buffer''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|1uM TSO.r04||||||||||1ul | |||
|- | |||
|dH20||||||||||7.625ul (6.525 + 1.1ul 2M NaCl) | |||
|- | |||
|10x RnaseIII buffer||||||||||0.5ul | |||
|- | |||
|ATP||||||||||0.625ul | |||
|- | |||
|0.5U PAP||||||||||0.25ul | |||
|- | |||
|} |
Revision as of 23:53, 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:
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
- Ambion PAP (0.4U) with 3'dATP
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
- Commercial mix Ambion (see above)
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
- 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
- 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 -RT (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- totoRNAseq mix -RT +NaCl (below)
- 10mM ATP
- 1mM ATP
- 1mM ATP/0.1mM 3' d-ATP
- 1mM ATP/0.5mM 3' d-ATP
- Commercial mix Ambion (see above)
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer
- totoRNAseq Reaction Buffer
Volume 1uM TSO.r04 1ul dH20 7.625ul (6.525 + 1.1ul 2M NaCl) 10x RnaseIII buffer 0.5ul ATP 0.625ul 0.5U PAP 0.25ul