User:Karmella Haynes/Notebook/Polycomb project/2010/10/04

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10/04/10

  • ✓ ChIP qPCR: KAH126-1
  • ✓ Western blot: continued from 10/03/10



ChIP qPCR
> Repeat from 10/04/10 using more template DNA > Set up each reaction in triplicate > Templates (use 2.0 μL, 8 rxns each):

  • KAH126-1 Input, pos (1:1)
  • KAH126-1 αmyc IP, exp (1:1)
  • KAH126-1 αIgG IP, neg (1:25)
  • 0 template (dH2O)

> Primers (12 rxns each):

  • INKARF D
  • INKARF E
  • INKARF F
  • INKARF G
  • MMP12 A
  • MMP12 B
  • MMP12 C
  • GAPDH B

--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O


Reagent 1 rxn x3 Primer mix (x13) DNA mix (x3 x9)
ChIP DNA (1:1) 2.0 6.0 --- 54.0
SYBR Green mix 7.5 22.5 97.5 ---
750 nM primers 3.0 9.0 39.0 ---
dH2O 2.5 7.5 --- 67.5
  15.0

--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 13.5 DNA mix to primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 10 min.
  • [95°C/ 10 sec, 58°C/ 10 sec, 72°C/ 10 sec] x45
  • Melt curve: 95°C/ 10 sec, 57°C -> 95°C/ 0.5 °C per step


> Conclusions: Results not as clear as previous. Try original PCR conditions and 4.5 uL template for primer optimization of full set (INKARF, MMP12, TNF)