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

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10/19/10

  • ✓ ChIP qPCR
  • ✓ HEK Gal4EED: luciferase assays



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. MMP12 A3
  2. MMP12 B2

--> Plate 2

  1. MMP12 C2
  2. MMP12 D3

--> Plate 3

  1. GAPDH A3
  2. GAPDH B2

--> 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

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