User:Karmella Haynes/Notebook/Polycomb project/2010/09/28

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09/28/10

  • ✓ RT-PCR: biological replicates for 4-day dox induction of Pc-ATF subset



RT-PCR
> Pc-ATF biological replicates (30 total)
--> Templates (6/15, 6/18, 6/23/10)

  1. KAH126-1 -dox
  2. KAH126-1 +dox
  3. KAH126-3 -dox
  4. KAH126-3 +dox
  5. KAH132-8 -dox
  6. KAH132-8 +dox
  7. KAH154-2 -dox
  8. KAH154-2 +dox
  9. FTRx -dox
  10. FTRx +dox

--> Primers

  1. p16INK 7C (94 bp), (0.5 μL 1:1 cDNA) (30 rxns)
  2. GAPDH 21A (100 bp), (0.5 μL 1:1000 cDNA) (30 rxns)
  3. MMP12 31A (127 bp), (5.0 μL 1:100 cDNA) (30 rxns)
  4. mCh (100 bp), (1.0 μL 1:1000 cDNA) (30 rxns)
  5. THPO 33A (103 bp)
  6. TNF 34A (119 bp)


Reagent p16, GAPDH mCh MMP12, THPO, TNF
cDNA 0.5 1.0 5.0
10 μM primers 1.0 1.0 1.0
2x GoTaq Green 10.0 10.0 10.0
dH2O 8.5 8.0 3.5
  20.0 μL


--> 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 092810 gel1.tif

> Conclusions: Looks like MMP12 is induced just by the KAH126 proteins. Cool! Repeat MMP12 and p16 with 2x more template