User:Karmella Haynes/Notebook/Polycomb project/2010/09/08

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09/08/10

 * &#x2713; RT-PCR: repeat mCherry for Nanostring-RNA cDNA set
 * &#x2713; p16 induction time course: set up KAH126-1, 154-2, 132-8, and FTRx in 10 cm plates (1:10 split, plain medium)
 * &#x2713; Cell culture: split confluent back-up stocks

RT-PCR > Use cDNA from Nanostring samples > Same PCR conditions as p16 (see 8/26/10) > Templates + Primers:
 * 1) Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1:500 cDNA dln.)

--> Add 2.5 μL cDNA (1:1000) --> PCR (96-well)* (*Same as p16INK PCR, 8/26/10)
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> Conclusions: Non-specific amplification again. Try again with both mCh1 and mCh2 primers and use less template DNA

Trial 2 > Templates + Primers:
 * 1) Nanostring (Ns.) sample set (1-20) + mCherry f1/r1 (1.0μL of 1:1000 cDNA dln.)
 * 2) Nanostring (Ns.) sample set (1-20) + mCherry f2/r2 (1.0μL of 1:1000 cDNA dln.)

--> Add 1.0 μL cDNA (1:1000) --> PCR (96-well)*
 * 95°C/ 3 min.
 * [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec.] x32
 * 72°C/ 3 min.
 * 4°C/ ∞



> Conclusions: Success! Both work. Use mCh1 for figure.


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