User:Karmella Haynes/Notebook/Polycomb project/2011/04/18

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04/18/11

 * &#x2713; ChIP qPCR: Pc-TF plates ch2, ch3
 * &#x2713; Transfection: repeat 4/12/11 expt. (11 days after dox w/o)
 * &#x2713; PCR: test luc primers

ChIP qPCR > See 12/20/11 > Set up each reaction 4x

>Plate ch2 --> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): --> 750 nM primers (24 rxns per primer pair):
 * 128-8.3 (40) input, pos
 * 128-8.3 (41) myc IP, uk
 * 128-8.3 (42) IgG IP, neg
 * 129-4 (43) input, pos
 * 129-4 (44) myc IP, uk
 * 129-4 (45) IgG IP, neg
 * 1) ATOH1 A1
 * 2) ATOH1 B3
 * 3) ATOH1 C2
 * 4) ATOH1 D1

>Plate ch3 --> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL): --> 750 nM primers (12 rxns per primer pair):
 * FTRx (29) input, pos
 * FTRx (38) myc IP, uk
 * FTRx (39) IgG IP, neg
 * 1) ATOH1 A1
 * 2) ATOH1 B3
 * 3) ATOH1 C2
 * 4) ATOH1 D1

--> Aliquot 52.0 primer mix into 1st well of each 4x set --> Add 8.0 (2.0 x4) DNA to 52.0 primer mix --> Aliquot 15.0 rxn mix to other 3 wells in each 4x set

Bio-Rad CFX96 qPCR (Kirschner lab) --> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C) --> 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

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 (4 days, wash-out day 11) --> Note: setting up double plates 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 (4x master mix = 20 μL) --> Serial dilutions: start w/ 8x DNA + dH2O = 40 μL --> Use 20 μL for serial dilutions > Fugene master mixes (x4, 24 total): 7.2 μL Fugene + 72.8 μL Opti-MEM --> R.T/ 5 min. > Add 20 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

PCR: test luc primers > Test new luc primers on HEK-luc cDNA > Templates (5 rxns ea.) > Primers (10 μM)
 * 1) 2/mock (#16) 1:10
 * 2) 2/mock (#16) 1:100
 * 3) 2/mock (#16) 1:1000
 * 1) luc 1 (110 bp)
 * 2) luc 2 (98 bp)
 * 3) luc 3 (117 bp)
 * 4) luc 4 (105 bp)
 * 5) GAPDH 21A (100 bp)

Bio-RAD 96-well
 * 95°C/ 3 min.
 * [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x30
 * 72°C/ 3 min.



> Conclusions: Primer pairs luc1, luc2, and luc4 work well. Use 1:100 dilution for subsequent qRT-PCR.


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