User:Karmella Haynes/Notebook/Polycomb project/2011/03/28

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03/28/11

  • ✓ qRT-PCR: Pc-TF -/+ dox plates 9, 10, 11
  • ✓ Cell culture: split 126-1, 129-4



qRT-PCR

> Plate 9
--> 750 nM Primers (& cDNA dilution)

  1. CASP10 41A (1:10)
  2. EOMES 42A (1:10)
  3. OTX 43D (1:10)
  4. SST 45C (1:10)

> Templates, use 2 μL

  1. KAH126-1, 2/21/10
  2. KAH126-1 +dox, 2/21/10
  3. KAH128-8, 2/21/10
  4. KAH128-8 +dox, 2/21/10
  5. KAH129-4, 2/21/10
  6. KAH129-4 +dox, 2/21/10
  7. FTRx, 6/15/10
  8. FTRx +dox, 6/15/10


> Plate 10
--> 750 nM Primers (& cDNA dilution)

  1. ATOH1 46D (1:10)
  2. TNF 34A (1:10)

> Templates, use 2 μL

  1. KAH126-1, 2/21/10
  2. KAH126-1 +dox, 2/21/10
  3. KAH128-8, 2/21/10
  4. KAH128-8 +dox, 2/21/10
  5. KAH129-4, 2/21/10
  6. KAH129-4 +dox, 2/21/10
  7. FTRx, 6/15/10
  8. FTRx +dox, 6/15/10


> Plate 11
--> 750 nM Primers (& cDNA dilution)

  1. CASP10 41A (1:10)
  2. EOMES 42A (1:10)
  3. OTX 43D (1:10)
  4. SST 45C (1:10)

> Templates, use 2 μL

  1. KAH126-1, 7/26/10 (use 1 μL, + 1 μL H2O)
  2. KAH126-1 +dox, 7/26/10 (use 1 μL, + 1 μL H2O), running low
  3. KAH128-8.3, 7/26/10
  4. KAH128-8.3 +dox, 7/26/10
  5. KAH129-4, 7/26/10
  6. KAH129-4 +dox, 7/26/10
  7. 132-8, 7/26/10
  8. 132-8 +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