Blue:RNA-Seq Experiments:10232013: Difference between revisions
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== | == Poly A Limiting Tests == | ||
:'''3'dATP Blocking''' | |||
*Test: | *Test: | ||
**Ambion PAP | **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/0mM 3'ATP | ||
***1mM ATP/0.01mM 3'ATP | ***1mM ATP/0.01mM 3'ATP | ||
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== Adaptation to totoRNAseq == | |||
:'''totoRNAseq Reaction Buffer''' | :'''totoRNAseq Reaction Buffer''' | ||
*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 | |||
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|- | |- | ||
|0.5U PAP||||||||||1ul | |0.5U PAP||||||||||1ul | ||
|- | |- | ||
|} | |} |
Revision as of 19:30, 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 (see above)
- totoRNAseq mix with 1mM ATP
- totoRNAseq mix with 10mM ATP
- Commercial mix pre-heat inactivated
- Ambion PAP vs Enzymatics PAP in RT/RNaseIII 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