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====gdna==== **H1: 240ng/ul x 100ul 175.5ng/ul Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit {| {{table}} | ||H1||1034s |- | RNA||3||43 |- | 10X Dnase I Buffer||5||5 |- | Rnase-Free Dnase I||2||2 |- | H2O||40||0 |- | |} 37C 10min Add 4 volumes of RNA binding buffer; Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s; Add 200ul RNA Wash buffer, spin @15k rpm for 30s; Add 10ul DNase/RNase-free water, spin @15k rpm for 30s; Yield: H1: 310.6ng/ul x 10ul 1034s: 336.4ng/ul x 10ul Reverse transcription {| {{table}} | ||H1||1034s |- | RNA||8||8 |- | 10mM dNTP||1||1 |- | 50uM dT12-18||1||1 |- | |} 65C 5min -> chill on ice for 1min Add H1 1034s 10X RT buffer 2 2 25mM MgC12 4 4 0.1M DTT 2 2 RnaseOUT 1 1 Superscript III 1 1 Incubate all tube at 50C for 50min. 2nd strand synthesis saved under Alan>2ss Add 5X 2nd strand buffer: 30ul 10mM dNTP 3ul DNA Pol I(10U/ul) 4ul RNase H(2U/ul) 1ul H2O 92ul 16C 2h -> 70C 10min -> add 2ul RNase I -> 37C 30min Purified with MinElute columns. Yield: H1: 252.5ng/ul x 15ul 1034s: 285.8ng/ul x 15ul Sample volume TBE Buffer 6X Loading Dye Total Volume /4 H1+gDNA 14.73 16 28 58.73 14.68 1034+cDNA 27.75 4 28 59.75 14.94
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