User:Karmella Haynes/Notebook/Polycomb project/2010/09/29

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


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09/29/10

 * &#x2713; HEK Gal4EED cells: died (neglected cells for too long, restart culture); request vial from Mirra
 * &#x2713; ChIP-PCR: all α-myc and α-IgG IP's

ChIP PCR > Note: dx = DMA/CH2O double x-linked; fx = CH2O x-linked --> Templates (use 1 μL and 2 μL 1:50 dilutions of each template; 20 rxn's total) --> Primers
 * 1) (8) 130-4 α-myc IP
 * 2) (10) 130-4 α-IgG IP
 * 3) (3) 126-1 dx α-myc IP
 * 4) (5) 126-1 dx α-IgG IP
 * 5) (18) 126-1 fx α-myc IP
 * 6) (20) 126-1 fx α-IgG IP
 * 7) (13) 132-8 dx α-myc IP
 * 8) (15) 132-8 dx α-IgG IP
 * 9) (23) 132-8 fx α-myc IP
 * 10) (25) 132-8 fx α-IgG IP
 * 1) INKARF D (135 bp) (20 rxns)
 * 2) GAPDH B (103 bp) (20 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:
 * Double x-linked samples are very non-specific; do not use
 * Single x-linked samples show mostly no pull-down for GAPDH neg ctrl.
 * 126-1 appears to IP with INKARF D
 * Next ChIP-PCR: compare antibody IP vs. supernatant to get ratios of enrichment before running qPCR


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