User:Karmella Haynes/Notebook/Polycomb project/2010/10/16

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10/16/10

  • ✓ ChIP qPCR: test primers
  • ✓ HepG2 Pc-ATF transfections: expand 1/2 transfected cells to 2.0 μg/mL puromycin 10 cm plates
  • ✓ HEK Gal4EED induction: split cells into new plates (1:2), keep old plate, refresh dox, add dox to 1-day samples; assay tomorrow



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 4.0 μL, 39 rxns each):

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

> Primers (6 rxns each):

  1. INKARF D1
  2. INKARF D2
  3. INKARF D3
  4. INKARF D4
  5. INKARF E1
  6. INKARF E2
  7. INKARF F1
  8. INKARF F2
  9. INKARF G1
  10. INKARF G2
  11. INKARF G3
  12. GAPDH A2
  13. GAPDH A3
  14. GAPDH B1
  15. GAPDH B2
  16. GAPDH C1

--> 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
INKARF D1 29.71 81.0/164.34 0 0 good
INKARF D2 32.02 74/241.25 0 0 C(t) too high
INKARF D3 26.32 77.5/288.45 0 0 good
INKARF D4 25.10 79.0/241.25 0 0 good
INKARF E1 31.06 72.5/282.19 0 0 good
INKARF E2 29.79 73.5/241.25 0 0 good
INKARF F1 29.67 83.0/253.75 37.17 76.0/163.70 good
INKARF F2 0 0 0 0 failed
INKARF G1 28.00 79.5/285.37 40.42 79.5/0 good
INKARF G2 29.56 81.5/204.49 0 0 good
INKARF G3 29.48 79.0/157.59 0 0 good
GAPDH A2 23.17 80.0/308.22 0 0 good
GAPDH A3 27.79 0 0 0 good
GAPDH B1 26.56 84.5/253.75 0 0 good
GAPDH B2 29.94 0 0 0 good
GAPDH C1 26.39 88.0/137.71 0 0 good


> Conclusions: Looks like using proper primer concentration improved the C(t) values. Use the following for qPCR (C(t) = 27 - 30)
> Continue primer test tomorrow