User:Karmella Haynes/Notebook/Polycomb project/2010/05/03

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05/03/10

  • ✓ RT-PCR: 4-day dox Pc-ATF lines
  • ✓ Bact. cultures: FlpE (4)
  • ✓ Senescence assay cultures: expand/split confluent cultures
  • ✓ Pc-ATF negative ctrl lines: Pick colonies (96-well plates, 32 colonies/line)


RT-PCR

> Continued from 4/29/10: semi-qRT-PCR
--> Use 1:100 dilution for mCh primer (10,000 might be too low)
--> 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. p15INK(5); 1:1
  2. p16INK4a(7B); 1:1
  3. mCh f1/r1; 1:100
  4. GAPDH(21A); 1:10,000
Reagent Volume Master mix (x20) RT-PCR 5/03/10
15 μL/lane; 2% agarose
cDNA 0.5 ---
10μM primers 1.0 10.0
2x GoTaq Green 10.0 100.0
dH2O 8.5 85.0
  20.0 μL
 
 
 
 
 
 
 
 


--> Looks interesting; 126-1 appears to increase p16INK
--> Redo GAPDH controls (21A and 21B primers) with 1:1000 dilution



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