Blue:RNA-Seq Experiments:11182013: Difference between revisions
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>B1lake |
>B1lake |
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Line 11: | Line 11: | ||
#TSO (no PAP) | #TSO (no PAP) | ||
#TSO + PAP | #TSO + Affy PAP | ||
#TSO + PAP + 3'dATP | #TSO + Affy PAP + 3'dATP | ||
#TSO + PAP + dATP | #TSO + Affy PAP + dATP | ||
#TSO + PAP + dNTP (similar conc as RT reaction) | #TSO + Affy PAP + dNTP (similar conc as RT reaction) | ||
#TSO + RNaseIII + PAP | #TSO + RNaseIII + Affy PAP | ||
#TSO + RNaseIII + PAP (no Rnase Inhibitor) | #TSO + RNaseIII + Affy PAP (no Rnase Inhibitor) | ||
Line 40: | Line 40: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|10x RNaseIII | |10x RNaseIII buffer||||||||||0.2ul | ||
|- | |- | ||
|RNase III||||||||||0.1ul | |RNase III or dH2O||||||||||0.1ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.7ul | ||
|- | |- | ||
|Total||||||||||2ul | |Total||||||||||2ul | ||
Line 63: | Line 63: | ||
|TSO||||||||||2ul | |TSO||||||||||2ul | ||
|- | |- | ||
|5x RT buffer | |5x RT buffer||||||||||0.5ul | ||
|- | |- | ||
|1mM ATP (or mix)||||||||||0.5ul | |1mM ATP (or mix)||||||||||0.5ul | ||
|- | |- | ||
| | |60U/ul Affymetrix PAP[1:10]||||||||||0.2ul | ||
|- | |- | ||
|40U/ul Rnase Inhibitor||||||||||0.2ul | |40U/ul Rnase Inhibitor||||||||||0.2ul | ||
|- | |- | ||
|dH2O||||||||||0. | |dH2O||||||||||0.2ul | ||
|- | |- | ||
|dNTP mix||||||||||0.4ul | |dNTP mix or dH2O||||||||||0.4ul | ||
|- | |- | ||
|Total||||||||||4ul | |Total||||||||||4ul |
Revision as of 22:40, 21 November 2013
Purpose
- Test Affymetrix PAP on TSO poly(A) tailing using similar protocol as 11-13-2013
- Test ability of [dATP] in the RT mix for inhibition of PAP activity
- Test whether RNaseIII retains activity on Poly(A) tailed RNA
RNAseIII-Fragmented Library Preparation
- Conditions
- TSO (no PAP)
- TSO + Affy PAP
- TSO + Affy PAP + 3'dATP
- TSO + Affy PAP + dATP
- TSO + Affy PAP + dNTP (similar conc as RT reaction)
- TSO + RNaseIII + Affy PAP
- TSO + RNaseIII + Affy PAP (no Rnase Inhibitor)
- RNA
Volume 1uM TSO.r04 1ul Total 1ul
- Incubate @ 72C 3 min
- Incubate @ 37C 1 min
Volume 10x RNaseIII buffer 0.2ul RNase III or dH2O 0.1ul dH20 0.7ul Total 2ul
- Incubate @ 37C 5 min
- 3' Tailing
Volume TSO 2ul 5x RT buffer 0.5ul 1mM ATP (or mix) 0.5ul 60U/ul Affymetrix PAP[1:10] 0.2ul 40U/ul Rnase Inhibitor 0.2ul dH2O 0.2ul dNTP mix or dH2O 0.4ul Total 4ul
- Incubate @ 37C 10 min