User:Karmella Haynes/Notebook/Polycomb project/2010/07/02

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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07/02/10

 * &#x2713; RT-PCR: end-point PCR on new cDNA set (for repeat of p16 activation expt.)
 * &#x2713; Western: protein prep for 129-4
 * &#x2713; Senescence assay: refresh medium, rotenone/DMSO (flow cytometry tomorrow)

RT-PCR

> See 4/29/10 semi-qRT-PCR --> cDNA templates (3 samples each)
 * 1) KAH126-1
 * 2) KAH126-1 +dox
 * 3) KAH126-3
 * 4) KAH126-3 +dox
 * 5) KAH132-8
 * 6) KAH132-8 +dox
 * 7) KAH154-2
 * 8) KAH154-2 +dox
 * 9) FTRx
 * 10) FTRx +dox

--> Primers; cDNA dilution
 * 1) p16INK4a(7B); 1:1
 * 2) GAPDH(21A); 1:1,000

--> Aliquot 19.5 μL of each DNA mix to appro. wells --> Add 0.5 cDNA to each well --> PCR (96-well)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 20 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Result: Non-specific bands looks very bad compared to last successful trail (see 5/03/10). Could the problem be too much RNA for cDNA synthesis? Try again using less RNA.


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