User:Karmella Haynes/Notebook/Polycomb project/2010/02/21

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02/21/10

  • ✓ Plan RT-PCR for 2/22/10

RT-PCR
--> Primers: (Based on RT-PCR trials from 11/5 and 11/9)

  1. MYT1 (1D), 1:1 cDNA
  2. p15INK (5), 1:1 cDNA
  3. RUNX3 (8A), 1:1 cDNA
  4. GAPDH loading control (21A), 1:10,000 cDNA

--> cDNA templates
Note: 130-2 not included because of plating accident. Will test later.

  1. KAH126-1
  2. KAH126-1 dox (4 days)
  3. KAH127-4
  4. KAH127-4 dox (4 days)
  5. KAH128-8
  6. KAH128-8 dox (4 days)
  7. KAH129-4
  8. KAH129-4 dox (4 days)
  9. KAH131-9
  10. KAH131-9 dox (4 days)
  11. KAH132-8
  12. KAH132-8 dox (4 days)
  13. KAH133-1
  14. KAH133-1 dox (4 days)
  15. FTRx
  16. FTRx dox (4 days)
  17. EZH2 k.d. (8 days)
  18. nt k.d. (8 days)


> PCR plate set-up

Row Primer pair Columns 1-12
A MYT1 (1D) cDNA templates 1-12
B MYT1 (1D) cDNA templates 13-18
C p15INK (5) cDNA templates 1-12
D p15INK (5) cDNA templates 13-18
E RUNX3 (8A) cDNA templates 1-12
F RUNX3 (8A) cDNA templates 13-18
G GAPDH (21A) cDNA templates 1-12
H GAPDH (21A) cDNA templates 13-18


> Master mixes

Reagent Volume Primer mixes (x19)
cDNA 0.5 ---
10μM primers 1.0 19.0 (19 x 1.0)
2x GoTaq Green 10.0 190.0 (19 x 10.0)
dH2O 8.5 161.5 (19 x 8.5)
  20.0 μL

--> Note: Dilute EZH2 k.d. templates 2x before use --> Aliquot 5.0 μL of each primer mix to appro. wells
--> Aliquot 0.5 μL of cDNA to appro. wells
--> PCR (96-well)

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

RT-PCR 2/22/10

--> Repeat experiment with full primer set. Next time, normalize loading based on GAPDH results from this gel


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