User:Karmella Haynes/Notebook/Polycomb project/2011/04/19

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


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04/19/11

 * &#x2713; qRT-PCR: Opt plate for HOXC4 and IGFBP2
 * &#x2713; qRT-PCR: Pc-TF plates 25, 26

qRT-PCR

> Optimization #3 --> See 4/3/11 for end-point PCR gel --> 750 nM Primers (& cDNA dilution)
 * 1-4. HOXC4 50A-D (1:10)
 * 5-8. IGFBP2 51A-D (1:10)

> Templates, use 2 μL
 * 1) KAH126-1 +dox (3/10/11)
 * 2) no template

--> Aliquot 39.0 primer mix into 1st well of each 3x set --> Add 6.0 (2.0 x3) DNA or water 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 (pick primers with C(t) closest to 30 for comparable amplification)
 * HOXC4: HOXC4 50B, lowest C(t) = 30.33; all gave C(t) of around 30, nice clean melt curve peaks, no zero template background
 * IGFBP2: IGFBP2 51A, highest C(t) = 24.00; all gave C(t) of around 23, nice clean melt curve peaks, no zero template background

> Plate 25 --> 750 nM Primers (& cDNA dilution)
 * 1) HOXC4 50B (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) KAH154-2 +dox "
 * 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) HOXC4 50B (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


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