User:Karmella Haynes/Notebook/Polycomb project/2010/10/06

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


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10/06/10

 * &#x2713; Order oligos: new primers for MMP12 and TNF ChIP-qPCR
 * &#x2713; Cell culture: split HEK Gal4-EED's; set up dox-induced Gal4-EED time course plate (12-well, ~50,000 cells/well)
 * &#x2713; ChIP/ co-IP: Protein A Sepahrose-H3K27me3 IP

Oligos
 * 1) MMP12 C f2; gaattgcaagcagtggtgctg
 * 2) MMP12 C r2; cagaccgtccccatacaatc
 * 3) MMP12 C f3; gcagattcttgctgatctgttc
 * 4) MMP12 C r3; ctcacagcagaataaccagc
 * 5) MMP12 D f1; cataacagtccttcaaaacc
 * 6) MMP12 D r1; tggtctaggaggctgtctgc
 * 7) MMP12 D f2; ctgagatgactgagggtaaag
 * 8) MMP12 D r2; ggagttcacttctgttatgg
 * 9) TNF C f2; gagaagcaactacagacc
 * 10) TNF C r2; cgtgggtcagtatgtgagag
 * 11) TNF C f3; ccagatgagctcatgggtttc
 * 12) TNF C r3; gctgggtgtgccaacaactg
 * 13) TNF D f1; gtgaaagatgtgcgctgatag
 * 14) TNF D r1; cagacatcctgtctctccatc
 * 15) TNF D f2; catggaaggtgctcactaag
 * 16) TNF D r2; ccacatctgtctccatatc

ChIP co-IP optimization > Not much luck with endogenous protein IP using Qingqing's protocol > Try simplified approach: --> Bind antibody with chromatin overnight --> Bind Protein A* beads with complexes for 5 hours Note: *H3K27me3 ab 07-449 was Protein A purified (Millipore) --> Wash with complete sonication buffer (same as IP using myc-conjugated beads) --> Elute with Western loading buffer

Today: > Try... > Prepare complete sonication buffer (see ###) > Prep 200 μL Protein A sepharose by washing and resuspending (3 times) in complete sonication buffer > Antibody binding --> Use H3K27me3 07-449 --> Use double x-linked KAH126-1 (surplus chromatin, not important)
 * chromatin + antibody, then beads and
 * beads + antibody, then chromatin

--> Rotate at 4°C/ overnight


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