User:Karmella Haynes/Notebook/Polycomb project/2010/02/21

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


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02/21/10

 * &#x2713; Plan RT-PCR for 2/22/10

RT-PCR --> Primers: (Based on RT-PCR trials from 11/5 and 11/9)
 * 1) MYT1 (1D), 1:1 cDNA
 * 2) p15INK (5), 1:1 cDNA
 * 3) RUNX3 (8A), 1:1 cDNA
 * 4) GAPDH loading control (21A), 1:10,000 cDNA

--> cDNA templates Note: 130-2 not included because of plating accident. Will test later.
 * 1) KAH126-1
 * 2) KAH126-1 dox (4 days)
 * 3) KAH127-4
 * 4) KAH127-4 dox (4 days)
 * 5) KAH128-8
 * 6) KAH128-8 dox (4 days)
 * 7) KAH129-4
 * 8) KAH129-4 dox (4 days)
 * 9) KAH131-9
 * 10) KAH131-9 dox (4 days)
 * 11) KAH132-8
 * 12) KAH132-8 dox (4 days)
 * 13) KAH133-1
 * 14) KAH133-1 dox (4 days)
 * 15) FTRx
 * 16) FTRx dox (4 days)
 * 17) EZH2 k.d. (8 days)
 * 18) nt k.d. (8 days)

> PCR plate set-up

> Master mixes

--> Note: Dilute EZH2 k.d. templates 2x before use --> Aliquot 5.0 μL of each primer mix to appro. wells --> Aliquot 0.5 μL of cDNA to appro. wells --> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞



--> Repeat experiment with full primer set. Next time, normalize loading based on GAPDH results from this gel


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