User:Karmella Haynes/Notebook/Polycomb project/2011/04/03

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04/03/11

  • ✓ RT-PCR: test new primers
  • ✓ Repressed luc reactivation: expand HEK293 23;4;9 to 12 plates total (split each current one 1:3); dox induction (4x no dox, 4x 2-day, 4x 4-day)
  • ✓ Pc-TF cell cultures: split all cultures; 6-well plate dox induction (4 days) for 129-4, 154-2, 156-5, 157-1 (3 reps ea.)



RT-PCR
> Test new RT-PCR primers on KAH126-1 (20 rxns total)
> Primers:
1-4. HOXC4 50A-D (86, 87, 86, 85 bp)
5-8. IGFBP2 51A-D (117, 120, 113, 114 bp)
9-12. NFE2 52A-D (90, 88, 118, 120 bp)
13-16. NEUROG1 53A-D (105, 117, 92, 98 bp)
17-20. SOX1 54A-D (84, 80, 80, 102 bp)

> Template:

  1. KAH126-1 +dox (3/10/11) 1:10


Reagent 1 rxn Template mix (21x)
cDNA 0.5 10.5
10 μM primers 1.0 ---
2x GoTaq Green 10.0 210.0
dH2O 8.5 178.5
  20.0 μL

--> 1 template mix: aliquot 19.0 μL
--> Add 1.0 μL primers

--> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 15 sec., 58°C/ 15 sec., 72°C/ 15 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞


RT-PCR 4/03/11

--> Conclusions:

  1. HOXC4: qPCR test A-D
  2. IGFBP2: qPCR test A-D
  3. NFE2: failed
  4. NEUROG1: failed
  5. SOX1: failed

Next: Do qRT-PCR test with 1:10 dilution of 126-1 (+dox 3/10/11)



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