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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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12/21/10

 * &#x2713; ChIP qPCR: continued from 12/20/10
 * &#x2713; 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
 * &#x2713; 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): > Primers (48 rxns per primer pair): --> Plate 3 --> Plate 4 --> Plate 5 --> Plate 6
 * 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
 * 1) GAPDH A3
 * 2) GAPDH B2
 * 1) MMP12 A3
 * 2) MMP12 B2
 * 1) MMP12 C2
 * 2) MMP12 D3
 * 1) TNF A2
 * 2) TNF B2

--> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O

--> 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: "


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