Blue:RNA-Seq Experiments:11182013
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Purpose[edit]
- 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[edit]
- 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
- Run on Urea gel
- Results: keeps failing for some unknown reason... repeated with 600U/ul pap and still no shift up of the TSO.