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

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03/15/11

 * &#x2713; qRT-PCR: Pc-TF -/+ dox plate 2
 * &#x2713; 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

--> 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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