User:Karmella Haynes/Notebook/Polycomb project/2010/10/05

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10/05/10

  • ✓ ChIP qPCR: optimize primers



ChIP qPCR
> Optimization to make sure input gives low C(t) compared to no template ctrl.
> Set up each reaction in triplicate
> Templates (use 4.0 μL, 39 rxns each):

  • KAH126-1 Input, pos (1:1)
  • 0 template (dH2O)

> Primers (6 rxns each):

  1. INKARF D
  2. INKARF E
  3. INKARF F
  4. INKARF G
  5. MMP12 A
  6. MMP12 A 2 (new)
  7. MMP12 A 3 (new)
  8. MMP12 B
  9. MMP12 C
  10. TNF A (new)
  11. TNF B (new)
  12. TNF C (new)
  13. GAPDH B

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


Reagent 1 rxn Primer mix (x7)
ChIP DNA (1:1) 4.5 ---
SYBR Green mix 7.5 52.5
750 nM primers 3.0 21.0 ---
dH2O --- --- ---
  15.0

--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 13.5 DNA 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/ 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


Results (0 = signal below threshold):

Primer pair Input C(t) Peak melt temp/ave height no DNA C(t) Peak melt temp/ ave height Conclusion
INKARF D 29.76 80.5/113.4 37.49 0 good
INKARF E 29.92 72.0/155.5 36.18 0 good
INKARF F 28.30 83.0/155.5 34.38 variable/104.6 good
INKARF G 27.06 79.5/220.5 0 0 good
MMP12 A 33.22 72.0/93.0 41.69 72.5/114.93 bad (do not use)
MMP12 A2 0 0 0 0 failed (do not use)
MMP12 A3 32.76 74.5/186.9 0 0 good
MMP12 B 31.23 74.0/236.2 43.66 0 good
MMP12 C 33.55 76.5/108.34 35.0 76.5/143.34 signal < bg (do not use)
TNF A 36.37 79.5/110.6 0 0 okay (high Ct, but no bg)
TNF B 29.08 84.5/135.8 44.31 0 good
TNF C 25.00 82.5/183.1 38.04 78.0/90.81 good
GAPDH B 26.61 84.0/104.9 35.36 78.5/86.17 good


> Design and order new MMP12 (downstream of tss), MMP12 C, and TNF A primers
> Continue with ChIP qPCR for good primers


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