User:Karmella Haynes/Notebook/Polycomb project/2011/04/12

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04/12/11

  • ✓ Transfection: HEK rep luc + Pc-TF's (5 days after dox w/o)
  • ✓ qPCR: Pc-TF -/+dox plate 18



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 1a/b: no dox
--> Plate 2a/b: Dox-treated cells (2 days, wash-out day 5)
--> Plate 3a/b: Dox-treated cells (4 days, wash-out day 5)
--> Note: double up this time to have enough cells for RNA preps

> Plate cells: Cells are very confluent; Collect cells from 1 10cm plate, resuspend 1/2 of cells in 25 mL medium, aliquot 1 mL per well in 24-well plate
--> Also split back-up plates 1:4 to make 4 per treatment; discard extras


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 5 μL (6x master mix = 30 μL)
--> Serial dilutions: start w/ 12x DNA + dH2O = 60 μL --> Use 30 μL for serial dilutions
> Fugene master mixes (x6, 24 total): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min.
> Add 15 μL DNA mix to each Fugene mix --> R.T./ 20 min.
> Add 25 μL complexes to each well (1 ml med. each); Grow cells at 37°C
> Assay luc activity after 2 days



qRT-PCR

> Plate 18
--> 750 nM Primers (& cDNA dilution)

  1. CASP10 41A (1:10)
  2. EOMES 42A "
  3. OTX2 43D "
  4. SST 45C "
  5. ATOH1 46D "
  6. TNF 34A "
  7. GAPDH 21A (1:1000), FTRx -/+ dox 6/18/10 only

> 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 mixes 1-6 (x13) Primer mix 7 (x7)
diluted cDNA 2.0 --- ---
SYBR Green mix 7.5 97.5 52.5
750 nM primers 3.0 39.0 21.0
dH2O 2.5 32.5 17.5
  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



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