User:Karmella Haynes/Notebook/Polycomb project/2010/10/18

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10/18/10

 * &#x2713; HepG2: split cells 1:5 in 75 cm flasks
 * HEK Gal4EED: luciferase assay on hold (waiting for assay buffer)
 * &#x2713; HEK Gal4EED: Use one Gal4EED induction plate to set up 12-well transfection plates; split 2 wells over 12 wells; split remaining well over three wells in original plate
 * &#x2713; ChIP qPCR: KAH 126-1, 132-8, 130-4 IPs; INKARF D1, E2, F1, G3 primers
 * &#x2713; Transfection: Fugene transfection for Gal4EED-induced HEK cells

ChIP qPCR > Set up each reaction in triplicate > Templates (use 4.5 μL): > Primers (30 rxns each): --> Plate 1 --> Plate 2 --> 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) INKARF D1
 * 2) INKARF E2
 * 1) INKARF F1
 * 2) INKARF G3

--> 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

Transfections > HEK293 Gal4-EED 23;4;9 cells, dox induced > Use Fugene, 12-well format, 1.5 mL medium per well > 4 plates total
 * 1) 0 dox (no Gal4-EED induction)
 * 2) 48 hr. dox (2 days)
 * 3) 120 hr. dox (5 days)
 * 4) 168 hr. dox (7 days)

> Master mix (4x, 1 per plate): 6 μL Fugene + 194 μL Opti-MEM --> R.T./ 5 min. > Add 4x vol. DNA --> R.T./ 20 min. > Remove 0.5 mL medium from each well (decrease volume to improve transfection efficiency) > Add 50 μL DNA/Fugene mix to each well (1.5 ml medium/ well); Grow cells at 37&deg;C > Refresh ab-free medium after 5 hours > Check RFP/ luciferase after 18-24 hours


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