User:Karmella Haynes/Notebook/Polycomb project/2010/09/23

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09/23/10

  • ✓ RT-PCR: Repeat KAH154-2, 156-5, correct loading



RT-PCR
> Pc-ATF NS repeats; balance loading for KAH154-2 and 156-5 samples (see 8/26/10 for GAPDH RT-PCR)
--> Templates

  1. KAH154-2 -dox (0.5x)
  2. KAH154-2 +dox (4x)
  3. KAH156-5 -dox (0.5x)
  4. KAH156-5 +dox (2x)

--> Primers

  1. p16INK 7C (94 bp) (4 rxns)
  2. MMP12 31A (127 bp) (4 rxns)
  3. THPO 33A (103 bp) (4 rxns)
  4. TNF 34A (119 bp) (4 rxns)
  5. GAPDH 21A (100 bp), 1:1000 cDNA (4 rxns)
  6. mCh (100 bp), 1:500 cDNA (4 rxns)


Reagent Single rxn
cDNA 2.0 total vol
10 μM primers 1.0
2x GoTaq Green 10.0
dH2O 7.0
  20.0 μL

--> Use 0.5 2x diluted cDNA + 1.5 dH2O for KAH154-2 -dox
--> Use 2.0 cDNA for KAH154-2 +dox
--> Use 0.5 2x diluted cDNA + 1.5 dH2O for KAH156-5 -dox
--> Use 1.0 cDNA + 1.0 dH2O for KAH156-5 +dox

--> 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 092310 gel2.tif

> Conclusions:

  • p16, GAPDH, and mCh are good
  • Forgot to use 1:10 dilution for MMP12, THPO, and TNF; repeat using the same adjustments as above, but use the 1:10 dilutions as starting cDNA stocks

File:KAH 092310 gel3.tif