User:Karmella Haynes/Notebook/Polycomb project/2010/09/08

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09/08/10

  • ✓ RT-PCR: repeat mCherry for Nanostring-RNA cDNA set
  • ✓ p16 induction time course: set up KAH126-1, 154-2, 132-8, and FTRx in 10 cm plates (1:10 split, plain medium)
  • ✓ Cell culture: split confluent back-up stocks



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 (1:500 cDNA dln.)


Reagent 1-20, MM (x21)</u>
cDNA ---
10 μM primers 21.0 (mCh f1/r1)
2x GoTaq Green 210.0
dH2O 136.5
  20.0 μL/rxn


--> Add 2.5 μL cDNA (1:1000)
--> 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)

File:KAH 090810 gel1.tif

> Conclusions: Non-specific amplification again. Try again with both mCh1 and mCh2 primers and use less template DNA


Trial 2
> Templates + Primers:

  1. Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1.0μL of 1:1000 cDNA dln.)
  2. Nanostring (Ns.) sample set (1-20) + mCherry f2/r2 (1.0μL of 1:1000 cDNA dln.)


Reagent 1-20, MM (x21)</u>
cDNA ---
10 μM primers 21.0 (mCh f1/r1)
2x GoTaq Green 210.0
dH2O 189.0
  20.0 μL/rxn


--> Add 1.0 μL cDNA (1:1000)
--> 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/ ∞

File:KAH 090810 gel2.tif File:KAH 090810 gel3.tif

> Conclusions: Success! Both work. Use mCh1 for figure.