User:Karmella Haynes/Notebook/Polycomb project/2010/10/08

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

10/08/10

  • ✓ ChIP qPCR: 126-1 & 132-8 continued
  • ✓ ChIP qPCR: primer test for new primer pairs



ChIP qPCR
> Set up each reaction in triplicate
> Templates (use 4.0 μL, 12 rxns each):

  1. KAH126-1 Input (#16), pos
  2. KAH126-1 αmyc IP (#18), unk
  3. KAH126-1 αIgG IP (#20), neg
  4. 0 template (dH2O)
  5. KAH132-8 Input (#21), pos
  6. KAH132-8 αmyc IP (#23), unk
  7. KAH132-8 αIgG IP (#25), neg
  8. 0 template (dH2O)

> Primers (24 rxns each):

  1. INKARF G
  2. MMP12 A3
  3. MMP12 B
  4. TNF A

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


Reagent 1 rxn Primer mix (x25)
ChIP DNA (1:1) 4.5 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
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



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):

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

> Primers (6 rxns each):

  1. MMP12 C2
  2. MMP12 C3
  3. MMP12 D1
  4. MMP12 D2
  5. TNF C2
  6. TNF C3
  7. TNF D1
  8. TNF D2

--> 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
MMP12 C2 31.47 79.5/181.04 0 0 good
MMP12 C3 40.46 75.5/123.24 0 0 failed, C(t) too high (do not use)
MMP12 D1 0 0 0 0 failed (do not use)
MMP12 D2 42.60 0 0 0 failed, C(t) too low (do not use)
TNF C2 31.61 81.5/151.35 41.69 72.5/114.93 good
TNF C3 31.30 80.5/230.28 0 0 good
TNF D1 40.32 78.5/108.5 0 0 failed, C(t) too low (do not use)
TNF D2 43.89 0 0 0 failed, C(t) too low (do not use)


> Conclusions:

  • Use MMP12 C2, TNF C3 for future experiments (TNF C3 overlaps with TNF C1)
  • Design new primers for MMP12 and TNF D regions



Personal tools