User:Karmella Haynes/Notebook/Polycomb project/2010/12/06

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12/06/10

  • ✓ ChIP qPCR 1: H3K27me3 IP set; optimize template load
  • ✓ ChIP qPCR 2: H3K27me3 IP set; INKARF primers
  • ✓ In vitro H3 tail binding assay: order H3K9me3 peptide and streptavidin beads
  • ✓ Cell culture: thaw KAH126-1, 128-8.3, 129-4, 130-4 for histone peptide binding assays
  • ✓ HepG2 TREx: change medium to DMEM plain (blast slowing cell growth?)



ChIP qPCR 1
> Set up each reaction in triplicate
> Templates (use 1, 2, 4.5 μL):

  • FTRx dx input, pos (9 wells)
  • FTRx dx H3K27me3 IP, uk (9)
  • FTRx dx myc, neg (9)
  • FTRx fx input, pos (9) *
  • FTRx fx H3K27me3 IP, uk (9) *
  • FTRx fx myc, neg (9)
  • 0 template (3)

Note: Accidentally switched fx input and K27 > Primers (57 rxns total):
--> Plate 1

  1. GAPDH B2

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


Reagent 1 rxn Primer mix (x60)
ChIP DNA up to 4.5 ---
SYBR Green mix 7.5 450.0
750 nM primers 3.0 180.0
dH2O --- ---
  15.0

--> Aliquot 31.5 primer mix into 1st well of each triplicate
--> Add 13.5 (4.5 x 3) DNA+dH2O to 31.5 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x 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

> Conclusion: GAPDH all volumes give C(t) = 27 for all input samples. 2 μL gives C(t) of about 33 - 35 for others. Melt curve peaks = 88.0 for all samples. Peak heights below threshold for K27me3 and myc IP as expected. Use 2 μL for future experimental samples.



ChIP qPCR 2
> Set up each reaction in triplicate
> Templates (use 2.0 μL):

  • FTRx dx input, pos
  • FTRx dx H3K27me3 IP, uk
  • FTRx dx myc, neg
  • FTRx fx input, pos
  • FTRx fx H3K27me3 IP, uk
  • FTRx fx myc, neg
  • 0 template

> Primers (21 rxns pre primer pair):
--> Plate 2

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

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


Reagent 1 rxn Primer mix (x23)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 172.5
750 nM primers 3.0 69.0
dH2O 2.5 57.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each triplicate
--> Add 6.0 (2.0 x 3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x 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