User:Karmella Haynes/Notebook/Polycomb project/2011/03/14

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03/14/11

  • ✓ qRT-PCR: Pc-TF -/+ dox plate 1



qRT-PCR plate 1
> 750 nM Primers (& cDNA dilution)

  1. mCh1 (1:1000)
  2. ACTC1 35A (1:10)
  3. GRHL2 37C (1:10)
  4. GAPDH 21A (1:1000)

> Templates, use 2 μL

  1. KAH126-1, 7/26/10
  2. KAH126-1 +dox, 7/26/10
  3. KAH154-2 +dox, 7/26/10 (accidentally switched loading)
  4. KAH154-2, 7/26/10 (accidentally switched loading)
  5. KAH128-8.3, 7/26/10
  6. KAH128-8.3 +dox, 7/26/10
  7. KAH157-1, 7/26/10
  8. KAH157-1 +dox, 7/26/10


Reagent 1 rxn Primer mix (x25)
diluted cDNA 2.0 ---
SYBR Green mix 7.5 187.5
750 nM primers 3.0 75.0
dH2O 2.5 62.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 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, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step