User:Karmella Haynes/Notebook/Polycomb project/2010/12/08

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12/08/10 ✓ ChIP qPCR: re-do FTRx fx INKARF F1 ChIP qPCR > Set up each reaction in triplicate > Templates (use 2.0 μL, duplicate in., K28, & myc): FTRx fx input, pos FTRx fx H3K27me3 IP, uk FTRx fx myc, neg FTRx fx input, pos FTRx fx H3K27me3 IP, uk FTRx fx myc, neg 0 template > Primers (21 rxns pre primer pair): --> Plate 2 INKARF F1 --> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O Reagent 1 rxn Primer mix (x23) ChIP DNA 2.0 --- SYBR Green mix 7.5 172.5 750 nM primers 3.0 69.0 dH2O 2.5 57.5   15.0 --> Aliquot 39.0 primer mix into 1st well of each triplicate --> Add 6.0 (2.0 x 3) DNA to 39.0 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