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

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10/18/10

  • ✓ HepG2: split cells 1:5 in 75 cm flasks
  • HEK Gal4EED: luciferase assay on hold (waiting for assay buffer)
  • ✓ 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
  • ✓ ChIP qPCR: KAH 126-1, 132-8, 130-4 IPs; INKARF D1, E2, F1, G3 primers
  • ✓ Transfection: Fugene transfection for Gal4EED-induced HEK cells



ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 4.5 μL):

  • 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)

> Primers (30 rxns each):
--> Plate 1

  1. INKARF D1
  2. INKARF E2

--> Plate 2

  1. INKARF F1
  2. INKARF G3

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


Reagent 1 rxn Primer mix (x31)
ChIP DNA (1:1) 4.5 ---
SYBR Green mix 7.5 232.5
750 nM primers 3.0 93.0
dH2O --- ---
  15.0

--> 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)


Wells Plasmid DNA Volume Fugene Opti-MEM
1. KAH126/MV2 500 ng 1.0 μL 1.5 μL 48.5 μL
2. KAH154/MV2 " 2.0 μL " "
3. KAH128/MV2 " 1.0 μL " "
4. KAH157/MV2 " 2.0 μL " "
5. KAH129/MV2 " 1.0 μL " "
6. KAH156/MV2 " 2.0 μL " "
7. KAH130/MV2 " 1.5 μL " "
8. KAH158/MV2 " 1.5 μL " "
9. KAH132/MV2 " 1.0 μL " "
10. KAH60/pcVN " 1.0 μL " "
11. no DNA --- --- " "

> 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°C
> Refresh ab-free medium after 5 hours
> Check RFP/ luciferase after 18-24 hours