User:Karmella Haynes/Notebook/Polycomb project/2011/04/21

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

04/21/11

  • ✓ Luc assay: HEK-luc rep +Pc-TF transfections (plate 2A)
  • ✓ ChIP qPCR: Plates ch4



ChIP qPCR
> First check for enrichment of H3K27me3 at putative target genes > Set up each reaction 4x

>Plate ch4
--> Templates (single x-linked chromatin; DNA prep no. in parenthesis; use 2.0 μL):

  • FTRx (29) input, pos
  • FTRx (38) H3K27me3 IP, uk
  • FTRx (39) IgG IP, neg

--> 750 nM primers (12 rxns per primer pair):

  1. NPPA A1
  2. NPPA B2
  3. NPPA C2
  4. NPPA D1
  5. EOMES A1
  6. EOMES B2
  7. EOMES C3
  8. EOMES D3



Reagent 1 rxn Primer mix (x13)
ChIP DNA 2.0 ---
SYBR Green mix 7.5 97.5
750 nM primers 3.0 39.0
dH2O 2.5 32.5
  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