User:Karmella Haynes/Notebook/Polycomb project/2010/12/07

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12/07/10

  • ✓ ChIP qPCR: continue with MMP12 primers
  • ✓ Cell culture: expand KAH126-1, 128-8.3, 129 to 75 cm flasks (130-4 did not grow)


ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 2.0 μL):

  • FTRx dx input, pos
  • FTRx dx H3K27me3 IP, uk
  • FTRx dx myc, neg
  • FTRx fx input, pos
  • FTRx fx H3K27me3 IP, uk
  • FTRx fx myc, neg
  • 0 template

> Primers (21 rxns pre primer pair):
--> Plate 2

  1. MMP12 A3
  2. MMP12 B2
  3. MMP12 C2
  4. MMP12 D3

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


Reagent 1 rxn Primer mix (x23)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 172.5
750 nM primers 3.0 69.0
dH2O 2.5 57.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each triplicate
--> Add 6.0 (2.0 x 3) DNA to 39.0 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



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