User:Karmella Haynes/Notebook/Polycomb project/2011/04/26

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04/26/11

  • ✓ ChIP qPCR: plates ch6, ch7, ch8, ch9



ChIP qPCR
> NPPA and EOMES showed H3K27me3 enrichment; check for Pc-TF enrichment > Set up each reaction 4x

>Templates
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  1. 132-8 (21) input, pos
  2. 132-8 (36) myc IP, uk
  3. 132-8 (37) IgG IP, neg
  4. 126-1 (16) input, pos
  5. 126-1 (32) myc IP, uk
  6. 126-1 (33) IgG IP, neg
  7. 128-8.3 (40) input, pos
  8. 128-8.3 (41) myc IP, uk
  9. 128-8.3 (42) IgG IP, neg
  10. 129-4 (43) input, pos
  11. 129-4 (44) myc IP, uk
  12. 129-4 (45) IgG IP, neg

> Plate ch6 primers
--> 750 nM primers (48 rxns per primer pair):

  1. EOMES A1
  2. EOMES B2

> Plate ch7 primers
--> 750 nM primers (48 rxns per primer pair):

  1. EOMES C3
  2. EOMES D3

> Plate ch8 primers
--> 750 nM primers (48 rxns per primer pair):

  1. NPPA B2
  2. NPPA C2

> Plate ch9 primers
--> 750 nM primers (48 rxns per primer pair):

  1. NPPA A1
  2. NPPA D1


Reagent 1 rxn Primer mix (x49)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 367.5
750 nM primers 3.0 147.0
dH2O 2.5 122.5
  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)
--> Note: Use increased annealing temp compared to previous ChIP PCR's (new primers optimized for 58°C)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step



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