User:Karmella Haynes/Notebook/Polycomb project/2010/10/17

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10/17/10

  • ✓ ChIP qPCR: continue primer test



ChIP qPCR primer test
> Optimization to make sure input gives low C(t) compared to no template ctrl.
> Set up each reaction in triplicate
> Templates (use 4.5 μL):

  • KAH132-8 Input, pos (1:1)
  • 0 template (dH2O)

> Primers (6 rxns each):

  1. GAPDH C2
  2. MMP12 A3
  3. MMP12 B1
  4. MMP12 B2
  5. MMP12 B3
  6. MMP12 C2
  7. MMP12 C4
  8. MMP12 D3
  9. MMP12 D4
  10. TNF A1
  11. TNF A2
  12. TNF B1
  13. TNF B2
  14. TNF C1
  15. TNF C4
  16. TNF D3

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


Reagent 1 rxn Primer mix (x6)
ChIP DNA (1:1) 4.5 ---
SYBR Green mix 7.5 45.0
750 nM primers 3.0 18.0
dH2O --- ---
  15.0

--> Make primer mix in 1st well of each set of six wells (two triplicates) --> Aliquot 31.5 primer mix into 1st well second triplicate
--> Add 13.5 (4.5 x 3) DNA to 31.5 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


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
GAPDH C2 25.10 83.0/177.05 0 0 good
MMP12 A3 27.85 74.5/316.35 0 0 good
MMP12 B1 31.76 74.5/285.35 0 0 C(t) too high
MMP12 B2 30.30 74.5/227.92 43.65 0 good
MMP12 B3 32.22 76.5/311.28 0 0 C(t) too high
MMP12 C2 25.15 79.5/309.15 0 0 good
MMP12 C4 24.64 78.0/283.42 0 0 good
MMP12 D3 28.84 81.5/190.51 0 0 good
MMP12 D4 29.57 82.0/227.39 0 0 good
TNF A1 32.4 80.0/218.15 44.17 0 C(t) too high
TNF A2 27.26 79.5/208.05 0 0 good
TNF B1 28.36 84.5/208.75 0 0 good
TNF B2 28.23 76.5/219.38 0 0 good
TNF C1 24.70 83.0/254.64 0 0 good
TNF C4 25.53 82.5/196.48 0 0 good
TNF D3 29.07 78.50/234.58 0 0 good


> Conclusions: Use primers with C(t) of 27-30

  • INKARF: D1, E2, F1, G3
  • GAPDH: A3, B2, C1
  • MMP12: A3, B2, C2, D3
  • TNF: A2, B2, C4, D3



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