Blue:RNA-Seq Experiments:06042014
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Quartz-Seq Library Preparation
RT Primer Removal
Volume Purified cDNA 3ul 10x PCR Buffer 0.4ul 10x Exo I buffer 0.2ul 100mM DTT 0.2ul Exonuclease I 0.6ul dH2O 1.6ul Total 6ul
- Takara Taq PCR Buffer:
Volume 1M Tris.HCl (pH 8.3) 100ul (100mM Final) 1M KCl 500ul (500mM Final) 1M MgCl2 15ul (15mM Final) dH2O 385ul Total 1ml
- Add directly to beads and mix
- Incubate @37C 30min
- Incubate @80C 20min
- Incubate @4C
Poly-A Tailing
Volume ExoI RXN 6ul 10x PCR Buffer 0.5ul 10mM dATP 1.5ul RNaseH 0.12ul TdT enzyme (Roche 03333566001) 0.42ul dH2O 2.46ul Total 11ul
- Incubate @37C 50 sec
- Incubate @65C 10min
- Incubate @4C
Second Strand Synthesis
Volume Poly-A Tailed Sample 11ul 2x Terra Direct PCR Buffer 25ul 10uM Tagging Primer 0.32ul Terra Polymerase 2ul dH2O 18.68ul Total 57ul
- Incubate @98C 2min 10 sec
- Incubate @40C 1min
- Incubate @68C 5min
- Transfer to ice
PCR Amplification
Volume 2nd Strand Sample 57ul 2x Terra Direct PCR Buffer 25ul 100uM PCR Primer 1ul dH2O 24ul Total 107ul
68C 1 sec 98C 10s -> 65C 15s -> 68C 5min 15x (1ng); 18x (100pg); 21x (10pg) 68C, 5min
Ran 5ul onto gel: