User:Karmella Haynes/Notebook/Polycomb project/2010/12/21

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12/21/10

  • ✓ ChIP qPCR: continued from 12/20/10
  • ✓ Colony growth assay: take bright field photos for 126-1 (~day 5); ~6.25x10^3 cells/ well showed good isolated colonies; will quantify cells per colony
  • ✓ ChIP: wash & elute 128-8, 129-4 (store at -20°C until after vacation)



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 per primer pair):
--> Plate 3

  1. GAPDH A3
  2. GAPDH B2

--> Plate 4

  1. MMP12 A3
  2. MMP12 B2

--> Plate 5

  1. MMP12 C2
  2. MMP12 D3

--> Plate 6

  1. TNF A2
  2. TNF B2

--> 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: wash & elute IP's
> Last ChIP qPCR showed higher than desired mock IP background. Try to reduce using more washes.
> Washes: Follow Casey's (modified) protocol for all samples; use 2x washes instead of one (see 12/01/10)
> Elution & DNA purification: Follow Qingqing's protocol (see 12/01/10). Stop after TE elution. Keep supernatant at -20°C until after vacation.
> 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: "


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