User:Karmella Haynes/Notebook/Polycomb project/2010/12/20

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12/20/10

  • ✓ ChIP: DNA purification day 3 (etOH ppt; resuspend in 500 10 mM Tris pH 8.0)
  • ✓ ChIP qPCR: new IP set and previous input samples
  • ✓ ChIP: myc/IgG IP's for 128-8.3 and 129-4 chromatin
  • ✓ Cell culture: expand thawed cultures 126-3, 154-2, 132-8, FTRx; split 126-1
  • ✓ HepG2 TREx: transfer from 6-well to 24-well plate to increase cell density (split into 2 wells)



ChIP qPCR
> Set up each reaction 4x
> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • 126-1 (16) input, pos
  • 126-1 (32) myc IP, uk
  • 126-1 (33) IgG IP, neg
  • 130-4 (06) input, pos
  • 130-4 (34) myc IP, uk
  • 130-4 (35) IgG IP, neg
  • 132-8 (21) input, pos
  • 132-8 (36) myc IP, uk
  • 132-8 (37) IgG IP, neg
  • FTRx (29) input, pos
  • FTRx (38) H3K27me3 IP, uk
  • FTRx (39) IgG IP, neg

> Primers (48 rxns pre primer pair):
--> Plate 2

  1. INKARF D1
  2. INKARF E2
  3. INKARF F1
  4. INKARF G3

--> 750 nM primer mix = 3 μL each 100 μM primer + 394 μL H2O


Reagent 1 rxn Primer mix (x50)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 375.0
750 nM primers 3.0 150.0
dH2O 2.5 125
  15.0

--> Aliquot 52.0 primer mix into 1st well of each 4x set
--> Add 8.0 (2.0 x4) DNA to 52.0 primer mix
--> Aliquot 15.0 rxn mix to other 3 wells in each 4x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 57°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 57°C -> 95°C/ 0.5°C per step



ChIP: 128-8.3, 129-4
> Samples (all form. x-linked chromatin)
1/2. 128-8: + 30 μL αmyc-beads (3400); + 30 μL mouse IgG-beads (3420)
3/4. 129-4: "
> Rotate @ 4°C/ overnight



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