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

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04/12/11

 * &#x2713; Transfection: HEK rep luc + Pc-TF's (5 days after dox w/o)
 * &#x2713; 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

> 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&deg;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

--> 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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