User:Karmella Haynes/Notebook/Polycomb project/2010/12/30

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12/30/10

  • ✓ ChIP qPCR: 128-8.3 and 129-4


ChIP qPCR
> Set up each reaction 4x
> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • 128-8 (40) input, pos
  • 128-8 (41) myc IP, uk
  • 128-8 (42) IgG IP, neg
  • 129-4 (43) input, pos
  • 129-4 (44) myc IP, uk
  • 129-4 (45) IgG IP, neg

> Primers (24 rxns per primer pair):
--> Plate 1

  1. GAPDH A3
  2. GAPDH B2
  3. INKARF D1
  4. INKARF E2

--> Plate 2

  1. INKARF F1
  2. INKARF G3
  3. MMP12 A3
  4. MMP12 B2

--> Plate 3

  1. MMP12 C2
  2. MMP12 D3
  3. TNF A2
  4. TNF B2

--> Plate 4

  1. TNF C4
  2. TNF D3


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


Reagent 1 rxn Primer mix (x26)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 195.0
750 nM primers 3.0 78.0
dH2O 2.5 65.0
  15.0

--> 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



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