Blue:RNA-Seq Experiments:10232013: Difference between revisions
Jump to navigation
Jump to search
>B1lake |
>B1lake |
||
Line 115: | Line 115: | ||
*Test: | *Test: | ||
**Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer | **Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer | ||
***Commercial mix (see above) | ***Commercial mix Ambion (see above) | ||
***totoRNAseq mix | ****10mM ATP | ||
***totoRNAseq mix | ****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 | |||
Line 127: | Line 138: | ||
|1uM TSO.r04||||||||||1ul | |1uM TSO.r04||||||||||1ul | ||
|- | |- | ||
|dH20|||||||||| | |dH20||||||||||6.4ul | ||
|- | |- | ||
|5x RT buffer||||||||||1.25ul | |5x RT buffer||||||||||1.25ul | ||
Line 135: | Line 146: | ||
|1mM ATP or 10mM ATP||||||||||0.625ul | |1mM ATP or 10mM ATP||||||||||0.625ul | ||
|- | |- | ||
|0.5U PAP|||||||||| | |0.5U PAP||||||||||0.25ul | ||
|- | |- | ||
|} | |} |
Revision as of 21:08, 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
- totoRNAseq Reaction Buffer
- 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
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