User:Karmella Haynes/Notebook/Polycomb project/2010/10/17

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10/17/10 ✓ ChIP qPCR: continue primer test ChIP qPCR primer test > Optimization to make sure input gives low C(t) compared to no template ctrl. > Set up each reaction in triplicate > Templates (use 4.5 μL): KAH132-8 Input, pos (1:1) 0 template (dH2O) > Primers (6 rxns each): GAPDH C2 MMP12 A3 MMP12 B1 MMP12 B2 MMP12 B3 MMP12 C2 MMP12 C4 MMP12 D3 MMP12 D4 TNF A1 TNF A2 TNF B1 TNF B2 TNF C1 TNF C4 TNF D3 --> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O Reagent 1 rxn Primer mix (x6) ChIP DNA (1:1) 4.5 --- SYBR Green mix 7.5 45.0 750 nM primers 3.0 18.0 dH2O --- ---   15.0 --> Make primer mix in 1st well of each set of six wells (two triplicates) --> Aliquot 31.5 primer mix into 1st well second triplicate --> Add 13.5 (4.5 x 3) DNA to 31.5 primer mix --> Aliquot 15.0 rxn mix to other 2 wells in each 3x set Bio-Rad CFX96 qPCR (Kirschner lab) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film 95°C/ 5 min. [95°C/ 15 sec, 57°C/ 15 sec, 72°C/ 15 sec] x45 Melt curve range 57°C -> 95°C/ 0.5°C per step Results (0 = signal below threshold): Primer pair Input C(t) Peak melt temp/ave height no DNA C(t) Peak melt temp/ ave height Conclusion GAPDH C2 25.10 83.0/177.05 0 0 good MMP12 A3 27.85 74.5/316.35 0 0 good MMP12 B1 31.76 74.5/285.35 0 0 C(t) too high MMP12 B2 30.30 74.5/227.92 43.65 0 good MMP12 B3 32.22 76.5/311.28 0 0 C(t) too high MMP12 C2 25.15 79.5/309.15 0 0 good MMP12 C4 24.64 78.0/283.42 0 0 good MMP12 D3 28.84 81.5/190.51 0 0 good MMP12 D4 29.57 82.0/227.39 0 0 good TNF A1 32.4 80.0/218.15 44.17 0 C(t) too high TNF A2 27.26 79.5/208.05 0 0 good TNF B1 28.36 84.5/208.75 0 0 good TNF B2 28.23 76.5/219.38 0 0 good TNF C1 24.70 83.0/254.64 0 0 good TNF C4 25.53 82.5/196.48 0 0 good TNF D3 29.07 78.50/234.58 0 0 good > Conclusions: Use primers with C(t) of 27-30 INKARF: D1, E2, F1, G3 GAPDH: A3, B2, C1 MMP12: A3, B2, C2, D3 TNF: A2, B2, C4, D3