User:Karmella Haynes/Notebook/Polycomb project/2010/09/20

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


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09/20/10

 * &#x2713; RT-PCR: full EZH2 kd set
 * &#x2713; ChIP PCR: Input vs. myc IP (partial set)
 * &#x2713; Cell culture: expand HepG2 cells; split HEK Gal4EED into selective maintenance (2.0 μg/mL puro) and non-selective media

RT-PCR > EZH kd set --> Templates --> Primers
 * 1) n.t. kd 1 8-day, 9/18/09
 * 2) EZH2 kd 1 8-day, 9/18/09
 * 3) n.t. kd 2 8-day, 9/18/09
 * 4) EZH2 kd 2 8-day, 9/18/09
 * 5) n.t. kd 4-day, 11/03/09
 * 6) EZH2 kd 4-day, 11/03/09
 * 7) n.t. kd 8-day, 11/08/09
 * 8) EZH2 kd 8-day, 11/08/09
 * 1) p16INK 7C (94 bp) (8 rxns)
 * 2) MMP12 31A (127 bp) (8 rxns)
 * 3) THPO 33A (103 bp) (8 rxns)
 * 4) TNF 34A (119 bp) (8 rxns)
 * 5) GAPDH 21A (100 bp), 1:1000 cDNA (8 rxns)
 * 6) EZH2 22A (71 bp) (8 rxns)

--> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> Conclusions:
 * p16INK: responds slightly, but still very repressed (probably due to DNA methylation)
 * MMP12: appears to respond to EZH2 kd. Need to quantitate using Image J.
 * THPO: may respond somewhat, pretty active overall.
 * TNF: appears to respond to EZH2 kd. Need to quantitate using Image J.
 * 8-day knockdown trial #2: Need to repeat using more nt kd cDNA (loading control signal too low for good comparison)
 * EZH2: Need to repeat with less template DNA; amplification not within linear range

ChIP PCR > Optimize template concentration > See 6/10/10 for successful primer test --> Templates --> Primers
 * 1) 126-1 Input
 * 2) 126-1 α-myc IP
 * 3) 130-4 Input
 * 4) 130-4 α-myc IP
 * 5) 132-8 Input
 * 6) 132-8 α-myc IP
 * 7) 126-1 Input (1:50)
 * 8) 126-1 α-myc IP (1:50)
 * 9) 130-4 Input (1:50)
 * 10) 130-4 α-myc IP (1:50)
 * 11) 132-8 Input (1:50)
 * 12) 132-8 α-myc IP (1:50)
 * 1) INKARF D (135 bp) (12 rxns)
 * 2) GAPDH A (103 bp) (12 rxns)

--> 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/ ∞



> Conclusions: GAPDH negative control comes down with 126-1 (double x-linked), slightly less with 132-8 (double x-linked) but not with 130-4 (single x-linked). As previously suspected, DMA plus formaldehyde may cause over-crosslinking. Continue only with single x-linked samples from now on.


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