User:Karmella Haynes/Notebook/Polycomb project/2011/03/13

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03/13/11

  • ✓ qRT-PCR: optimization
  • ✓ Luc activity assay: 4day Pc-TF, 12 days luc rep
  • ✓ Transfection: Pc-TF, 4-day luc-repressed cells



qRT-PCR optimization
> Optimize primers for qRT-PCR (use 750 nM)

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

> Template dilutions: KAH126-1 +dox, use 2 μL

  1. 1:10
  2. 1:100
  3. 1:1000
  4. 0 cDNA

750 nM primer mix =

  • 30 μL 10 μM primer mix + 370 μL H2O
  • 3 μL each 100 μM primer + 394 μL H2O


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

--> Results: C(t) Mean

  1. ACTC1 35A: 30.89 (1:10)
  2. GRHL2 37C: 39.46 (1:10)
  3. NPPA 40A: 36.68 (1:10)
  4. CDKN2A 7C: 39.36 (1:10)
  5. MMP12 31B: 38.33 (1:10)
  6. THPO 33A: 32.24 (1:10)
  7. mCh1: 24.73 (1:1000)
  8. GAPDH 21A: 22.43 (1:1000)
  • No-template control gave zero signal for all reactions
  • Conclusions: use 1:10 for experimental primers, 1:1000 for mCh and GAPDH



Luc Activity Assay

Luc activity assay > Cells: dox-induced Gal4-EED expression/ 12 days, followed by transfection for 4 days
1-6. KAH160/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
7-12. KAH170/MV1 (human Pc-TF), 0, 50, 100, 200, 400, 800 ng plasmid
> Sample processing:

  • Resuspend cells in 1 mL medium already in wells
  • Aliquot 3x 100 μL samples into 96-well plate


--> Luciferase not higher than neg. ctrl. Do not do cell counts (save Millipore tips for later experiments).
--> Conclusions: ability to reactivate does not appear to increase with longer Gal4-EED expression. Nest, 4-day transient Gal4-EED induction, dox wash-out, keep growing cells and try transfection every 2 days following



Fugene transfection

  1. KAH160: human Pc-TF
  2. KAH165: human PCD
  3. KAH161: fly Pc-TF
  4. KAH167: fly PCD
  5. KAH162: fish Pc-TF
  6. KAH166: fish PCD
  7. KAH170: deleted PCD
  8. mock (Fugene)


--> Plate 1: Dox-treated cells (4 days, wash-out day 0)
--> Plate 2: no dox

Wells Plasmid DNA Volume Fugene Opti-MEM
1-3 KAH160/MV1 100, 200, 400 ng 1.2 μL = 400 ng 1.8 μL 18.2 μL
4-6 KAH165/MV1 " 0.8 μL = 400 ng " "
7-9 KAH161/MV1 " 1.0 μL = 400 ng " "
10-12 KAH167/MV1 " 1.0 μL = 400 ng " "
13-15 KAH162/MV1 " 1.1 μL = 400 ng " "
16-18 KAH166/MV1 " 0.9 μL = 400 ng " "
19-21 KAH170/MV1 " 1.0 μL = 400 ng " "
22-24 mock " 0 " "

> Add DNA to stdH2O for a total volume of 10 μL (2x master mix = 20 μL)
--> Serial dilutions: 4x DNA + dH2O = 40 μL --> Use 20 μL for serial dilutions
> Fugene master mixes (x2, 24 total): 3.6 μL Fugene + 36.4 μL Opti-MEM --> R.T/ 5 min.
> Add 20 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 30 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days

> Note: Continue cultures w/ ~20% of the cells (4 mL of 10 mL resuspension) to seed new 10 cm plates (1 dox+, 1 dox-); for transfection plates, use remainder of cells (each brought up to 25 mL total)


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