User:Karmella Haynes/Notebook/Polycomb project/2011/04/19

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04/19/11

  • ✓ qRT-PCR: Opt plate for HOXC4 and IGFBP2
  • ✓ qRT-PCR: Pc-TF plates 25, 26


qRT-PCR

> Optimization #3
--> See 4/3/11 for end-point PCR gel
--> 750 nM Primers (& cDNA dilution)

  • 1-4. HOXC4 50A-D (1:10)
  • 5-8. IGFBP2 51A-D (1:10)

> Templates, use 2 μL

  1. KAH126-1 +dox (3/10/11)
  2. no template


Reagent 1 rxn Primer mix (x7)
diluted cDNA 2.0 ---
SYBR Green mix 7.5 52.5
750 nM primers 3.0 21.0
dH2O 2.5 17.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA or water to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step

> Results (pick primers with C(t) closest to 30 for comparable amplification)

  • HOXC4: HOXC4 50B, lowest C(t) = 30.33; all gave C(t) of around 30, nice clean melt curve peaks, no zero template background
  • IGFBP2: IGFBP2 51A, highest C(t) = 24.00; all gave C(t) of around 23, nice clean melt curve peaks, no zero template background


> Plate 25
--> 750 nM Primers (& cDNA dilution)

  1. HOXC4 50B (1:10)

--> Templates, use 2 μL

  1. KAH126-1 (2/21/10)
  2. KAH126-1 +dox "
  3. KAH126-1 (6/15/10)
  4. KAH126-1 +dox "
  5. KAH126-1 (7/26/10)
  6. (no template)
  7. KAH128-8 (2/21/10)
  8. (no template)
  9. KAH128-8.3 (7/26/10)
  10. KAH128-8.3 +dox "
  11. KAH129-4 (2/21/10)
  12. KAH129-4 +dox "
  13. KAH129-4 (7/26/10)
  14. KAH129-4 +dox "
  15. KAH154-2 (6/15/10)
  16. KAH154-2 +dox "
  17. KAH154-2 (7/26/10)
  18. KAH154-2 +dox "
  19. KAH154-2 (4/08/11 B)
  20. KAH154-2 +dox "
  21. KAH156-5 (7/26/10)
  22. KAH156-5 +dox "
  23. KAH156-5 (4/08/11 A)
  24. KAH156-5 +dox "
  25. KAH156-5 (4/08/11 B)
  26. KAH156-5 +dox "
  27. KAH157-1 (7/26/10)
  28. KAH157-1 +dox "
  29. KAH157-1 (4/08/11 A)
  30. KAH157-1 +dox "
  31. KAH157-1 (4/08/11 B)
  32. KAH157-1 +dox "

> Plate 25
--> 750 nM Primers (& cDNA dilution)

  1. HOXC4 50B (1:10)

--> Templates, use 2 μL

  1. KAH132-8 (6/15/10)
  2. KAH132-8 +dox "
  3. KAH132-8 (6/18/10)
  4. KAH132-8 +dox "
  5. KAH132-8 (6/23/10)
  6. KAH132-8 +dox "
  7. FTRx (2/21/10)
  8. FTRx +dox "
  9. FTRx (6/15/10)
  10. FTRx +dox "
  11. FTRx (6/18/10)
  12. FTRx +dox "
  13. FTRx (7/26/10)
  14. FTRx +dox "
Reagent 1 rxn Primer mix (x49), make 2 Primer mix (x43)
diluted cDNA 2.0 --- ---
SYBR Green mix 7.5 367.5 322.5
750 nM primers 3.0 147.0 129.0
dH2O 2.5 122.5 107.5
  15.0

--> Aliquot 39.0 primer mix into 1st well of each 3x set
--> Add 6.0 (2.0 x3) DNA to 39.0 primer mix
--> Aliquot 15.0 rxn mix to other 2 wells in each 3x set

Bio-Rad CFX96 qPCR (Kirschner lab)
--> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film

  • 95°C/ 5 min.
  • [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
  • Melt curve range 58°C -> 95°C/ 0.5°C per step


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