User:Karmella Haynes/Notebook/Polycomb project/2010/07/02

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07/02/10

  • ✓ RT-PCR: end-point PCR on new cDNA set (for repeat of p16 activation expt.)
  • ✓ Western: protein prep for 129-4
  • ✓ Senescence assay: refresh medium, rotenone/DMSO (flow cytometry tomorrow)


RT-PCR

> See 4/29/10 semi-qRT-PCR
--> cDNA templates (3 samples each)

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


--> Primers; cDNA dilution

  1. p16INK4a(7B); 1:1
  2. GAPDH(21A); 1:1,000
Reagent Volume Master mix (x31) RT-PCR 7/02/10
15 μL/lane; 2% agarose
cDNA 0.5 ---
10μM primers 1.0 31.0
2x GoTaq Green 10.0 310.0
dH2O 8.5 263.5
  20.0 μL
 
 
 
 
 
 
 
 

--> Aliquot 19.5 μL of each DNA mix to appro. wells --> Add 0.5 cDNA to each well --> PCR (96-well)

  • 95°C/ 3 min.
  • [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 20 sec.] x35
  • 72°C/ 3 min.
  • 4°C/ ∞


--> Result: Non-specific bands looks very bad compared to last successful trail (see 5/03/10). Could the problem be too much RNA for cDNA synthesis? Try again using less RNA.


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