User:Karmella Haynes/Notebook/Polycomb project/2011/04/20

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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04/20/11

 * &#x2713; qRT-PCR: Pc-TF plates 27, 28
 * &#x2713; cDNA: HEK-Gal4-luc Pc-TF RNA preps 1-8 (no silencing), 9-15 (2-day dox set, remainder), 17-24 (4-day dox set)
 * &#x2713; qRT-PCR: HEK-Gal4-luc Pc-TF cDNA plate HEK1

qRT-PCR

> Plate 27 --> 750 nM Primers (& cDNA dilution)
 * 1) IGFBP2 51A (1:10)

--> Templates, use 2 μL
 * 1) KAH126-1 (2/21/10)
 * 2) KAH126-1 +dox "
 * 3) KAH126-1 (6/15/10)
 * 4) KAH126-1 +dox "
 * 5) KAH126-1 (7/26/10)
 * 6) (no template)
 * 7) KAH128-8 (2/21/10)
 * 8) (no template)
 * 9) KAH128-8.3 (7/26/10)
 * 10) KAH128-8.3 +dox "
 * 11) KAH129-4 (2/21/10)
 * 12) KAH129-4 +dox "
 * 13) KAH129-4 (7/26/10)
 * 14) KAH129-4 +dox "
 * 15) KAH154-2 (6/15/10)
 * 16) KAH154-2 +dox "
 * 17) KAH154-2 (7/26/10)
 * 18) (no template)
 * 19) KAH154-2 (4/08/11 B)
 * 20) KAH154-2 +dox "
 * 21) KAH156-5 (7/26/10)
 * 22) KAH156-5 +dox "
 * 23) KAH156-5 (4/08/11 A)
 * 24) KAH156-5 +dox "
 * 25) KAH156-5 (4/08/11 B)
 * 26) KAH156-5 +dox "
 * 27) KAH157-1 (7/26/10)
 * 28) KAH157-1 +dox "
 * 29) KAH157-1 (4/08/11 A)
 * 30) KAH157-1 +dox "
 * 31) KAH157-1 (4/08/11 B)
 * 32) KAH157-1 +dox "

> Plate 25 --> 750 nM Primers (& cDNA dilution)
 * 1) IGFBP2 51A (1:10)

--> Templates, use 2 μL
 * 1) KAH132-8 (6/15/10)
 * 2) KAH132-8 +dox "
 * 3) KAH132-8 (6/18/10)
 * 4) KAH132-8 +dox "
 * 5) KAH132-8 (6/23/10)
 * 6) KAH132-8 +dox "
 * 7) FTRx (2/21/10)
 * 8) FTRx +dox "
 * 9) FTRx (6/15/10)
 * 10) FTRx +dox "
 * 11) FTRx (6/18/10)
 * 12) FTRx +dox "
 * 13) FTRx (7/26/10)
 * 14) FTRx +dox "

--> 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

cDNA synthesis --> Samples: Cells treated with dox for 0, 2, 4 days; transfected with Pc-TF/MV1 constructs 5 days after dox wash-out

> oligo(dT) Primer annealing --> Do 4 rxn's for 129-4 #1 --> 2 rxn's ea. for 129-4 +dox #4 & #5

> cDNA synthesis mix --> 16 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 0.8 μL RNase H, 37°C/ 20 min. --> Store at -20°C

qRT-PCR

> Plate HEK1 --> 750 nM Primers (& cDNA dilution)
 * 1) GAPDH 21A (1:100)
 * 2) mCh1 "
 * 3) luc1 "
 * 4) luc4 "

--> Templates, use 2 μL
 * 1) (17) 4dy dox/ 160
 * 2) (18) 4dy dox/ 165
 * 3) (19) 4dy dox/ 161
 * 4) (20) 4dy dox/ 167
 * 5) (21) 4dy dox/ 162
 * 6) (22) 4dy dox/ 166
 * 7) (23) 4dy dox/ 170
 * 8) (24) 4dy dox/ mock

--> 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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