User:Karmella Haynes/Notebook/Polycomb project/2010/10/11

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10/11/10

  • ✓ ChIP qPCR: 126-1, 132-8 continued
  • ✓ HepG2 culture: Split HepG2 for transfections; back-up stock 1:5
  • ✓ HEK Gal4EED culture: induce 12-well plate with dox (set 1); back-up stock 1:5



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. MMP12 C2
  2. TNF B
  3. TNF C3
  4. TNF C1

--> 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