User:Karmella Haynes/Notebook/Polycomb project/2011/03/23

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03/23/11

  • ✓ qRT-PCR: test new primers


qRT-PCR primer test
--> 750 nM Primers (& cDNA dilution); 30 μL 10 μM primer mix + 370 μLdH2O

  1. CASP10 41A
  2. CASP10 41D
  3. EOMES 42A
  4. EOMES 42D
  5. OTX2 43D
  6. SST 45C
  7. SST 45D
  8. ATOH1 46A
  9. ATOH1 46D

--> Templates, use 2 μL

  1. KAH126-1 +dox, 3/10/11, 1:10
  2. KAH126-1 +dox, 3/10/11, 1:100
  3. no cDNA


Reagent 1 rxn Template mix (x28)
diluted cDNA 2.0 56.0
SYBR Green mix 7.5 210.0
750 nM primers 3.0 ---
dH2O 2.5 70.0
  15.0

--> Aliquot 36.0 template mix into 1st well of each 3x set
--> Add 9.0 (3.0 x3) primers to 36.0 template 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

--> Results (Ave C(t), 1:10 cDNA)

  1. CASP10 41A: 32.75 (use this primer)
  2. CASP10 41D: 33.06
  3. EOMES 42A: 37.63 (use this primer)
  4. EOMES 42D: 42.23
  5. OTX2 43D: 33.63 (use this primer)
  6. SST 45C: 34.93 (use this primer)
  7. SST 45D: 36.85
  8. ATOH1 46A: 36.99
  9. ATOH1 46D: 33.44 (use this primer)



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