Haynes Lab:Notebook/Jan/2015/12/16

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RT-PCR for HOXD11/HOXD12/GAPDH on U2OS and iPS cells REACTION LIST   Template cDNA Gene Target Rxn 1: U2OS cells HOXD11 (1) Rxn 2: U2OS cells HOXD11 (2) Rxn 3: U2OS cells HOXD11 (3) Rxn 4: U2OS cells HOXD12 (1) Rxn 5: U2OS cells HOXD12 (2) Rxn 6: U2OS cells GAPD Rxn 7: iPS cells HOXD11 (1) Rxn 8: iPS cells HOXD11 (2) Rxn 9: iPS cells HOXD11 (3) Rxn 10: iPS cells HOXD12 (1) Rxn 11: iPS cells HOXD12 (2) Rxn 12: iPS cells GAPD Rxn 13: no template HOXD11 (1) Rxn 14: no template HOXD11 (2) Rxn 15: no template HOXD11 (3) Rxn 16: no template HOXD12 (1) Rxn 17: no template HOXD12 (2) Rxn 18: no template GAPD Plate Layout Primers HOXD11 (1): HOXD11 L1, HOXD11 R1, UPL Probe 18 HOXD11 (2): HOXD11 L2, HOXD11 R2, UPL Probe 60 HOXD11 (3): HOXD11 L3, HOXD11 R3, UPL Probe 1 HOXD12 (1): HOXD12 L1, HOXD12 R1, UPL Probe 70 HOXD12 (2): HOXD12 L2, HOXD12 R2, UPL Probe 84 GAPD: GAPD primers, GAPD probe Mixes PCR master mix for each gene target. One gene target is in 3 reactions, and 3 replicates per reaction, +1 for pipetting error. 3 x 3 + 1 = 10 Reagent (Single well) Gene Target (x10) GAPD (x10) 2x LC480 Probes Master (7.5 μL) 75.0 75.0 20 μM Forward primer (0.3 μL) 3.0 3.0 GAPD primers* 20 μM Reverse primer (0.3 μL) 3.0 --- 10 μM UPL probe (0.3 μL) 3.0 3.0 GAPD UPL probe* PCR H2O (0.1 μL) 1.0 4.0 Total vol. (8.5 μL) 85.0 85.0 Resulting 1.5 mL tubes: HOXD11 (1) - 85.0 μL HOXD11 (2) - 85.0 μL HOXD11 (3) - 85.0 μL HOXD12 (1) - 85.0 μL HOXD12 (2) - 85.0 μL GAPD - 85.0 μL Master mix for each template First, make a 1:10 dilution of the cDNA. One cell type is in 6 reactions, 3 replicates + 1 pipetting error. 6 * 3 + 1 = 19 Reagent (Single well) cDNA Template (x19) 1:10 cDNA dilution (2.0 μL) 38.0* PCR H2O (4.5 μL) 85.5 Total vol. (6.5 μL) 123.5 *For the no template control, use PCR H2O instead of cDNA. Resulting 1.5 mL tubes: U2OS - U2OS cells cDNA, 123.5 μL iPS - iPS cells cDNA, 123.5 μL Con - no template control, 123.5 μL