User:Karmella Haynes/Notebook/Polycomb project/2010/09/07

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09/07/10

  • ✓ RT-PCR: Pc-ATF Nanostring set, mCherry; test HHEX primers on KAH129-4
  • ✓ Sequencing order: KAH160-170/KAH015(FUW) for Casey (trouble expressing some constructs; check via sequencing)
  • ✓ Order oligos: new gene subset (see 9/05/10)



RT-PCR
> Use cDNA from Nanostring samples
> Same PCR conditions as p16 (see 8/26/10)
> Templates + Primers:

  1. Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1:100 cDNA dln.)
  2. Ns. KAH129-4 -/+ dox + HHEX 28A
  3. Ns. KAH129-4 -/+ dox + HHEX 28B


Reagent 1-20, MM (x20) 11-14, MM (x5)
cDNA --- ---
10 μM primers 20.0 (mCh f1/r1) ---
2x GoTaq Green 200.0 50.0
dH2O 80.0 42.5
  20.0 μL  


--> Add 5.0 μL cDNA (1:1000) to 1-20
--> Add 0.5 μL cDNA (1:1) and 1.0 μL primers to 11-14 --> PCR (96-well)*

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
  • 72°C/ 3 min.
  • 4°C/ ∞

(*Same as p16INK PCR, 8/26/10)

RT-PCR 9/07/10

> Conclusions: Non-specific products for mCherry (re-do with 2 uL template); HHEX gave no signal



Order oligos

  • IL4
  • MMP12
  • NPPA
  • THPO
  • TNF


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