User:Karmella Haynes/Notebook/Polycomb project/2010/12/22

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12/22/10

  • ✓ ChIP qPCR: continued



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

  • 126-1 (16) input, pos
  • 126-1 (32) myc IP, uk
  • 126-1 (33) IgG IP, neg
  • 130-4 (06) input, pos
  • 130-4 (34) myc IP, uk
  • 130-4 (35) IgG IP, neg
  • 132-8 (21) input, pos
  • 132-8 (36) myc IP, uk
  • 132-8 (37) IgG IP, neg
  • FTRx (29) input, pos
  • FTRx (38) H3K27me3 IP, uk
  • FTRx (39) IgG IP, neg

> Primers (48 rxns per primer pair):
--> Plate 7

  1. TNF C4
  2. TNF D3

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


Reagent 1 rxn Primer mix (x50)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 375.0
750 nM primers 3.0 150.0
dH2O 2.5 125
  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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