User:Karmella Haynes/Notebook/Polycomb project/2010/09/22

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09/22/10

 * &#x2713; ChIP PCR: test MMP12 and TNF primers on KAH130-4 IP
 * &#x2713; ChIP: IP for KAH126-1, 132-8 (form. x-linked chromatin)

ChIP PCR > KAH130-4 CH2O-x-linked chromatin --> Templates --> Primers
 * 1) 130-4 Input (1:50) (12 rxns)
 * 2) 130-4 α-myc IP (1:50) (12 rxns)
 * 3) 130-4 α-IgG IP (1:50) (12 rxns)
 * 1) INKARF C (138 bp)
 * 2) INKARF D (135 bp)
 * 3) INKARF E (86 bp)
 * 4) INKARF F (100 bp)
 * 5) MMP12 A (111 bp)
 * 6) MMP12 B (114 bp)
 * 7) MMP12 C (138 bp)
 * 8) TNF A (120 bp)
 * 9) TNF B (123 bp)
 * 10) TNF C (119 bp)
 * 11) GAPDH A (103 bp)
 * 12) GAPDH B (103 bp)

--> 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: Might be some pull-down on INKARFD; try again with full set of chromatin IP's using 1x and 2x amount of ChIP DNA

ChIP: myc-bead pull-down > Use new CH2O-x-linked chromatin preps > See 9/15/10 > Sonicated ~6 mL samples prepped according to Qingqing's protocol --> Add:
 * 720 μL 10% Triton X-100
 * 72 μL 10% Na-deoxycholate (DOC)
 * 500 μL TE (pH 8.0)
 * 6 μL 1000x PLAAC
 * 60 μL 100x PMSF

> Binding: --> Rotate at 4°C overnight --> Wash and elute tomorrow
 * 1) KAH126-1: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) KAH126-1: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
 * 3) KAH132-8: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 4) KAH132-8: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

9/23/10 > Wash & elute IP's (Keep everything on ice ) --> Prepare washing buffer (complete sonication buffer): --> Spin down beads at 3000 rpm/ 3 min./ 4°C --> Save 100 μL Supernatant. Discard remaining sup. --> Wash beads in 500 μL wash buffer (1 min.). Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup. Repeat three more times (4 washes total) --> Add 500 μL 1% SDS/TE buffer. Incubate at 65°C/ 15 min. Vortex every 2 min. --> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube.
 * 6 mL Buffer III
 * 60 μL 100x PMSF
 * 6 μL 1000x PLAAC
 * 720 μL 10% Triton X-100
 * 72 μL 10% Na-deoxycholate (DOC)
 * 500 μL TE (pH 8.0)

> Reverse crosslinks and digest protein --> Add 400 uL 1% SDS/TE buffer to 100 uL saved Supernatant. --> Thaw Input aliquot for each cell line. Add 400 uL 1% SDS/TE buffer to 100 uL Input. --> Samples:
 * 1) KAH126-1 input
 * 2) KAH126-1 α-myc Sup
 * 3) KAH126-1 α-myc IP
 * 4) KAH126-1 α-IgG Sup
 * 5) KAH126-1 α-IgG IP
 * 6) KAH132-8 input
 * 7) KAH132-8 α-myc Sup
 * 8) KAH132-8 α-myc IP
 * 9) KAH132-8 α-IgG Sup
 * 10) KAH132-8 α-IgG IP

--> Add 20 μL (20 μg) Pronase to each sample --> Incubate at 42°C/ 1 hr. --> Incubate at 65 °C/ 2 days


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