User:Karmella Haynes/Notebook/Polycomb project/2010/05/05

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05/05/10

  • ✓ FlpE: cultures (2 from 20 μg/mL Amp plate, 2 from 100 μg/mL Amp plate)
  • ✓ RT-PCR: Pc-ATF 4-day dox set, p14ARF & KRT17
  • ✓ Senescence assay cultures: Expand 132-4, -6 to 10 cm plates; KAH126-1 not growing well (freeze down current culture, PS7, on next passage); thaw fresh U2OS Flp-in T-REx


RT-PCR

> Continued from 5/04/10: semi-qRT-PCR
--> cDNA templates

  1. KAH126-1
  2. KAH126-1(+dox)
  3. KAH127-4
  4. KAH127-4(+dox)
  5. KAH128-8
  6. KAH128-8(+dox)
  7. KAH129-4
  8. KAH129-4(+dox)
  9. KAH130-2
  10. KAH130-2(+dox)
  11. KAH131-9
  12. KAH131-9(+dox)
  13. KAH132-8
  14. KAH132-8(+dox)
  15. KAH133-1
  16. KAH133-1(+dox)
  17. FTRx
  18. FTRx(+dox)

--> Primers; cDNA dilution

  1. p14ARF(6); 1:1
  2. KRT17(14A); 1:100


Reagent Volume Primer mix (x20) RT-PCR 5/05/10
15 μL/lane; 2% agarose
cDNA 0.5 ---
10μM primers 1.0 20.0
2x GoTaq Green 10.0 200.0
dH2O 8.5 170.0
  20.0 μL
 
 
 
 
 
 
 
 
 
 
 
 


--> Quantitate KRT17 using Image J



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