User:Karmella Haynes/Notebook/Polycomb project/2010/10/03

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10/03/10

  • ✓ ChIP qPCR: experimental sample set 126-1
  • ✓ Western: ChIP/ Co-IP for 126-1 (DsRed and H3K27me3)
  • ✓ Culture: split HepG2's 1:10


qPCR
> Set up each reaction in triplicate > Templates (use 4.5 μL):

  • KAH126-1 Input, pos (1:25)
  • KAH126-1 αmyc IP, exp (1:25)
  • KAH126-1 αIgG IP, neg (1:25)
  • 0 template (dH2O)

> Primers (12 rxns each):

  • INKARF D
  • INKARF E
  • INKARF F
  • INKARF G
  • MMP12 A
  • MMP12 B
  • MMP12 C
  • GAPDH B

--> 750 nM primer mix = 30 μL 10 μM mix + 470 μL H2O


Reagent 1 rxn x3 Primer mix (x13)
ChIP DNA (1:50) 4.5 12.5 ---
SYBR Green mix 7.5 22.5 97.5
750 nM primers 3.0 9.0 39.0
dH2O --- --- ---
  15.0

--> Aliquot 31.5 primer mix into 1st well of each triplicate set
--> Add 12.5 1:25 template DNA to primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in 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/ 3°C per step


> Conclusions:

  • C(t) still not very far from background. Try again using 2.0 μL 1:1 template


Western blot: ChIP/ Co-IP
> IP samples from 10/01/10; 30 μL each, ready to load
> Prep input and sup samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 15 min.

Gels 1 & 2

1. PageRuler Plus (10 μL)
2. KAH126-1 input
3. KAH126-1 α-myc sup
4. KAH126-1 α-myc IP
5. KAH126-1 α-IgG sup (neg ctrl)
6. KAH126-1 α-IgG IP (neg ctrl)
7. α-myc beads (neg ctrl)
8. PageRuler
9. dye
10. dye

Note: Use IP samples A or B for gel 1/2 (IP matched with appropriate sup)

10/03/10 Ponceau S stain
Ponceau S stained filters

> Block: 5% milk/PBST, 4°C/overnight


10/04/10
> Primary staining: 5% BSA/PBST, 4°C/o.n.

  1. rabbit α-DsRed 632496, 1:1000, 5 mL
  2. rabbit α-H3K27me3 07-449, 1:500, 5 mL


10/05/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.

  1. donkey α-rabbit-HRP, 1:5000; 5 mL
  2. donkey α-rabbit-HRP, mouse α-GAPDH-HRP 1:5000; 5 mL

> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)

  • KAH126-1: 43 kD
  • H3K27me3: 15 - 17 kD


Western blot 10/05/10
Conclusions:



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