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
  1. TSO (no PAP)
  2. TSO + Affy PAP
  3. TSO + Affy PAP + 3'dATP
  4. TSO + Affy PAP + dATP
  5. TSO + Affy PAP + dNTP (similar conc as RT reaction)
  6. TSO + RNaseIII + Affy PAP
  7. 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.