User:Karmella Haynes/Notebook/Polycomb project/2011/03/15

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03/15/11

  • ✓ qRT-PCR: Pc-TF -/+ dox plate 2
  • ✓ Luc activity assay



qRT-PCR plate 2
> 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. KAH129-4, 7/26/10
  2. KAH129-4 +dox, 7/26/10
  3. KAH156-5, 7/26/10
  4. KAH156-5 +dox, 7/26/10
  5. KAH132-8, 7/26/10
  6. KAH132-8 +dox, 7/26/10
  7. FTRx, 7/26/10
  8. FTRx +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



Luc Activity Assay

Luc activity assay > Cells: dox-induced Gal4-EED expression/ 4 days, followed by transfection for 2 days
1-3. KAH160/MV1 (human Pc-TF), 100, 200, 400 ng plasmid
4-6. KAH165/MV1 (human PCD)
7-9. KAH161/MV1 (fly Pc-TF)
10-12. KAH167/MV1 (fly PCD)
13-15. KAH163/MV1 (fish Pc-TF)
16-18. KAH166/MV1 (fish PCD)
19-21. KAH170/MV1 (VP64)
22-24. mock

> Sample processing:

  • Remove 500 μL medium; resuspend cells in remaining ~500μL medium
  • Aliquot 3x 100 μL samples into 96-well plate


--> Luc signal highest in 100 ng samples. Do cell counts on these.
--> Final experiment on cells immediately following Gal4-EED induction. Restart fresh HEK culture for subsequent experiments (next week)



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