User:Karmella Haynes/Notebook/Polycomb project/2010/12/21

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12/21/10 ✓ ChIP qPCR: continued from 12/20/10 ✓ Colony growth assay: take bright field photos for 126-1 (~day 5); ~6.25x10^3 cells/ well showed good isolated colonies; will quantify cells per colony ✓ ChIP: wash & elute 128-8, 129-4 (store at -20°C until after vacation) ChIP qPCR > Set up each reaction 4x > Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): 126-1 (16) input, pos 126-1 (32) myc IP, uk 126-1 (33) IgG IP, neg 130-4 (06) input, pos 130-4 (34) myc IP, uk 130-4 (35) IgG IP, neg 132-8 (21) input, pos 132-8 (36) myc IP, uk 132-8 (37) IgG IP, neg FTRx (29) input, pos FTRx (38) H3K27me3 IP, uk FTRx (39) IgG IP, neg > Primers (48 rxns per primer pair): --> Plate 3 GAPDH A3 GAPDH B2 --> Plate 4 MMP12 A3 MMP12 B2 --> Plate 5 MMP12 C2 MMP12 D3 --> Plate 6 TNF A2 TNF B2 --> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O Reagent 1 rxn Primer mix (x50) ChIP DNA 2.0 --- SYBR Green mix 7.5 375.0 750 nM primers 3.0 150.0 dH2O 2.5 125   15.0 --> Aliquot 52.0 primer mix into 1st well of each 4x set --> Add 8.0 (2.0 x4) DNA to 52.0 primer mix --> Aliquot 15.0 rxn mix to other 3 wells in each 4x 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 ChIP: wash & elute IP's > Last ChIP qPCR showed higher than desired mock IP background. Try to reduce using more washes. > Washes: Follow Casey's (modified) protocol for all samples; use 2x washes instead of one (see 12/01/10) > Elution & DNA purification: Follow Qingqing's protocol (see 12/01/10). Stop after TE elution. Keep supernatant at -20°C until after vacation. > Samples (all form. x-linked chromatin) 1/2. 128-8: + 30 μL αmyc-beads (3400); + 30 μL mouse IgG-beads (3420) 3/4. 129-4: "