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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

03/13/11

 * &#x2713; qRT-PCR: optimization
 * &#x2713; Luc activity assay: 4day Pc-TF, 12 days luc rep
 * &#x2713; 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
 * 1:10
 * 1:100
 * 1:1000
 * 1) 0 cDNA

750 nM primer mix =
 * 30 μL 10 μM primer mix + 370 μL H2O
 * 3 μL each 100 μM primer + 394 μL H2O

--> 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

> 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&deg;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)


 * }