User:Karmella Haynes/Notebook/Polycomb project/2011/04/07

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04/07/11

  • ✓ qRT-PCR: Pc-TF plate 17
  • ✓ RNA preps: 129-4, 154-2, 156-5, 157-1 -/+dox
  • ✓ Repr. luc reactivation expt.: wash out dox from 2 and 4-day plates; split all 1:2



qRT-PCR

> Plate 17
--> 750 nM Primers (& cDNA dilution)

  1. mCh1 (1:1000)
  2. GAPDH 21A (1:1000)
  3. ACTC1 35A (1:10)
  4. GRHL2 37C "
  5. NPPA 40A "
  6. CDKN2A 7C "
  7. MMP12 31B "
  8. THPO 33A "

> Templates, use 2 μL

  1. KAH132-8, 6/18/10
  2. KAH132-8 +dox, 6/18/10
  3. KAH132-8, 6/23/10
  4. KAH132-8 +dox, 6/23/10


Reagent 1 rxn Primer mix (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



RNA Preps
> Resuspend in 15 μL THE RNA storage solution; store at -80°C until cDNA synthesis