User:Karmella Haynes/Notebook/Polycomb project/2010/10/01

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10/01/10

  • ✓ ChIP/ Co-IP: IP for KAH126-1, 132-8, and 130-4 chromatin (for Western)
  • ✓ RT-PCR: Pc-ATF biological rep's, redo p16 (2x)
  • ✓ Cell culture: expand HEK Gal4-EED to two 75 cm2 flasks (2 μg/mL puro)


RT-PCR
> Pc-ATF biological replicates (30 total)
--> Templates (6/15, 6/18, 6/23/10)

  1. KAH126-1 -dox
  2. KAH126-1 +dox
  3. KAH126-3 -dox
  4. KAH126-3 +dox
  5. KAH132-8 -dox
  6. KAH132-8 +dox
  7. KAH154-2 -dox
  8. KAH154-2 +dox
  9. FTRx -dox
  10. FTRx +dox

--> Primers

  1. p16INK 7C (94 bp), (1.0 μL 1:1 cDNA) (30 rxns)



Reagent 1 rxn
cDNA 1.0
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 8.0
  20.0 μL


--> 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/ ∞


RT-PCR 10/01/10

> Conclusions:


ChIP: myc-bead pull-down
> See 9/15/10
> Sonicated ~6 mL samples prepped according to Qingqing's protocol
--> Added:

  • 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 (3 each):

  1. KAH130-4: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  2. KAH130-4: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
  3. KAH132-8: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  4. KAH132-8: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry
  5. KAH126-1: 400 μL chromatin + 10 μL mouse α-myc-bead (#3400) slurry
  6. KAH126-1: 400 μL chromatin + 10 μL mouse α-IgG-bead (#3420) slurry

--> Rotate at 4°C >overnight


10/03/10
> Wash & elute IP's (Keep everything on ice)
--> Prepare washing buffer (complete sonication buffer):

  • 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)

--> Spin down beads at 3000 rpm/ 3 min./ 4°C
--> Save 100 μL Supernatant. Discard remaining sup.
--> Wash beads in 400 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).

> α-myc bead control for Western: --> Add 400 μL wash buffer to 10 μL α-myc beads. Spin down beads at 3000 rpm/ 3 min./ 4°C. Discard sup.
--> Add 30 μL 1x loading dye (250 uL 4x l.d. + 750 dH2O, + ~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).



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