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

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08/17/10

 * &#x2713; 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: --> See 5/04/10 for successful p16INK RT-PCR
 * 1) p16INK 7A
 * 2) p16INK 7B
 * 3) p16INK 7C

--> 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.


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