User:Karmella Haynes/Notebook/Polycomb project/2010/08/17

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

08/17/10

  • ✓ RT-PCR: KAH126-1 -/+ dox cDNA



RT-PCR
> Need to re-optimize p16INK primers, 7B produces non-specific bands in biological replicates > KAH126-1 -/+ dox cDNA
1,2. KAH126-1 -/+ dox (6/15/10 Trizol, 5 μg RNA)
3,4. KAH126-1 -/+ dox (6/18/10 Trizol, 5 μg RNA)
5,6. KAH126-1 -/+ dox (7/26/10 RNeasy, 2 μg RNA)
7,8. FTRx -/+ rotenone (7/08/10 Trizol, 5 μg RNA)

> Primer pairs:

  1. p16INK 7A
  2. p16INK 7B
  3. p16INK 7C

--> See 5/04/10 for successful p16INK RT-PCR


Reagent 1:1 cDNA MM (x10) File:KAH 081710 PCR1.tif
15 μL/lane; 2% agarose
cDNA 0.5 (1:1) ---
10μM primers 1.0 10.0
2x GoTaq Green 10.0 100.0
dH2O 8.5 85.0
  20.0 μL
 

--> Aliquot 19.5 μL ("1:1" tubes) of each DNA mix to appro. wells
--> Add 0.5 (1:1) cDNA to each well
--> 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/ ∞


--> Conclusion: p16 7C gives the most specific product. Use this primer pair for all samples.