User:Karmella Haynes/Notebook/Polycomb project/2011/04/14

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04/14/11

  • ✓ qRT-PCR: Pc-TF -/+ dox plates 21, 22
  • ✓ Luc assay: Transfections from 4/12/11
  • ✓ RNA prep: Transfections from 4/12/11; lyse 3 wells in 1 mL Trizol and store at -80°C
  • ✓ Order oligos: luciferase reporter primers



qRT-PCR

> Plate 21
--> 750 nM Primers (& cDNA dilution)

  1. mCh1 (1:1000)
  2. GAPDH 21A (1:1000)
  3. ACTC1 35A (1:10)
  4. ATOH1 46D "
  5. CASP10 41A "
  6. CDKN2A 7C "
  7. EOMES 42A "
  8. GRHL2 37C "

> Templates, use 2 μL

  1. KAH156-5, 4/08/11 A
  2. KAH156-5 +dox, 4/08/11 A
  3. KAH156-5, 4/08/11 B
  4. KAH156-5 +dox, 4/08/11 B


> Plate 22
--> 750 nM Primers (& cDNA dilution)

  1. MMP12 31B (1:10)
  2. NPPA 40A "
  3. OTX2 43D "
  4. SST 45C "
  5. THPO 33A "
  6. TNF 34A "

> Templates, use 2 μL

  1. KAH156-5, 4/08/11 A
  2. KAH156-5 +dox, 4/08/11 A
  3. KAH156-5, 4/08/11 B
  4. KAH156-5 +dox, 4/08/11 B


Reagent 1 rxn Primer mixes 1-6 (x13)
diluted cDNA 2.0 ---
SYBR Green mix 7.5 97.5
750 nM primers 3.0 39.0
dH2O 2.5 32.5
  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



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