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Blue:RNA-Seq Experiments:11052013
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== Purpose == *Use bioanalyzer to test different fragmentation/poly(A) conditions == RNAseIII-Fragmented Library Preparation == :'''Conditions''' #No RNase III (5mM MnCl2)/no PAP treatment #RNaseIII (5mM MnCl2)/no PAP treatment #RNaseIII (5mM MgCl2)/no PAP treatment #RNaseIII (10mM MnCl2)/no PAP treatment #RNaseIII (15mM MnCl2)/no PAP treatment #RNaseIII (20mM MnCl2)/no PAP treatment #No RNase III (5mM MnCl2)/PAP (2.5mM MnCl2) #RNaseIII (5mM MnCl2)/PAP (2.5mM MnCl2) #RNaseIII (5mM MgCl2)/PAP (2.5mM MgCl2) #RNaseIII (10mM MnCl2)/PAP (5mM MnCl2) #RNaseIII (15mM MnCl2)/PAP (7.5mM MnCl2) #RNaseIII (20mM MnCl2)/PAP (10mM MnCl2) :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |UHRR 10ng/ul ||||||||||1ul |- |5x Ambion PAP buffer||||||||||0.08ul |- |dH20||||||||||0.09ul |- |Total||||||||||1.17ul |- |} :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1min'' :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA||||||||||1.17ul |- |MnCl2/MgCl2||||||||||0.4ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.33ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 5 min'' :::''Column Purify Samples 1-6'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |fRNA||||||||||2ul |- |5x Ambion PAP Buffer||||||||||0.72ul |- |ATP (or mix)||||||||||0.25ul |- |2U/ul PAP||||||||||0.2ul |- |40U/ul Rnase Inhibitor||||||||||0.2ul |- |dH2O||||||||||0.63ul |- |} :::''Incubate @ 37C 10 min'' :::''Column Purify Samples 7-12'' [[File:2100_expert_Eukaryote_Total_RNA_Pico_DE72903057_2013-11-06_12-24-52.pdf]] Results: *RNaseIII fragmentation is very efficient (fragments centered on about 75-100nt) and doesn't change with increasing MnCl2 *PAP is not fully complete, and smear is quite high. Seems to decrease in length with increasing MnCl2 *Substitution of MnCl2 for MgCl2 shows similar smear pattern as unfragmented - indicating MnCl2 works better for RNaseIII == Testing dATP on PAP Activity == #1mM ATP:0mM dATP (1:0) #1mM ATP:0.05mM dATP (1:20) #1mM ATP:0.1mM dATP (1:10) #1mM ATP:0.2mM dATP (1:5) #1mM ATP:1mM dATP (1:1) #0mM ATP:1mM dATP (0:1) 1ul 10uM TSO 1ul 5x ePAP buffer 0.5ul 25mM MnCl2 0.5ul ATP/dATP 1.0 ul 0.4U/ul ePAP 1ul dH2O [[File:11052013_gel1.jpg]] Results: dATP was able to limit ePAP activity to a small degree when added 1:1 with ATP
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