User:Karmella Haynes/Notebook/Polycomb project/2010/09/13

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09/13/10

 * &#x2713; RNA prep/ cDNA: p16 induction time course
 * &#x2713; ChIP/ Co-IP: KAH130-4 sample
 * &#x2713; Midipreps: Use QIAfilter Plasmid Midi kit (100 mL culture, used Maxi volumes to prep and ppt lysate, midi volumes to wash/ elute/ ppt DNA)
 * &#x2713; Culture: HEK cells failed to grow; get back-up vial from Mirra

RNA prep > Use Qiagen QIAshredder and RNeasy kit (no BME added to lysis buffer) > Elute with 50 μL RNase-free H2O

--> Store remainder of RNA at -80°C

7/26/10 > oligo(dT) Primer annealing

> cDNA synthesis mix --> 16 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 0.8 μL RNase H, 37°C/ 20 min. --> Store at -20°C

ChIP: myc-bead pull-down > See 6/29/10 > Prep sonicated ~6 mL samples according to Qingqing's protocol --> Add: --> Save 4x 100 μL samples as "Input"
 * 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) KAH130-4: 500 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
 * 2) KAH130-4: 500 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

9/14/10 > 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 50 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 60 μL 1x loading dye (50 uL 4x l.d. + 150 RIPA, ~50 mM DTT) to each pellet. Heat at 100°C/ 5 min., vortex. --> Clear supernatant by spinning at 4000 rpm/ 2 min/ RT. Transfer sup to new tube (save at -20°C for Western later).
 * 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)


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