User:Karmella Haynes/Notebook/Polycomb project/2010/10/19

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10/19/10

 * &#x2713; ChIP qPCR
 * &#x2713; HEK Gal4EED: luciferase assays

ChIP qPCR > Set up each reaction in triplicate > Templates (use 4.5 μL): > Primers (30 rxns each): --> Plate 1 --> Plate 2 --> Plate 3 --> 750 nM primer mix = 3 μL 100 μM each primer + 394 μL H2O
 * KAH126-1 fx Input, pos (1:1)
 * KAH126-1 fx myc IP, uk (1:1)
 * KAH126-1 fx IgG, neg (1:1)
 * KAH132-8 fx Input, pos (1:1)
 * KAH132-8 fx myc IP, uk (1:1)
 * KAH132-8 fx IgG, neg (1:1)
 * KAH130-4 fx Input, pos (1:1)
 * KAH130-4 fx myc IP, uk (1:1)
 * KAH130-4 fx IgG, neg (1:1)
 * 0 template, NTC (dH2O)
 * 1) MMP12 A3
 * 2) MMP12 B2
 * 1) MMP12 C2
 * 2) MMP12 D3
 * 1) GAPDH A3
 * 2) GAPDH B2

--> Aliquot 31.5 primer mix into 1st well of each 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

HEK Gal4-EED Luciferase assays > Transfection looks good (~50%) for 5 and 7 day induced cells > Very low transfection efficiency for 0 and 2 day induced cells > Apirate off old medium. Resuspend cells in 1 mL new medium. Collect 500 uL cells into epi tubes (use 100 uL for luc assay (triplicates) and rest for cell counts) > Re-plate remaining 500 uL in new plates. Bring medium vol up to 1.5 (non selective med.). > Luciferase assay: --> Plate 3 (5-day), samples 1-11* --> Plate 4 (7-day), samples 1-11* --> Gal4EED induction plate, samples 1-12 (3x 0 dox, 2 day, 5 day, 7 day) > *Note: transfection of Gal4-ATF (KAH60) showed very low RFP signal

Conclusions:
 * Expect to see low luc expression in dox-induced cells
 * Looking for increased luc expression in Pc-ATF cells, not in controls


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