User:Karmella Haynes/Notebook/Polycomb project/2010/09/14

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


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09/14/10

 * &#x2713; Midiprep check: KpnI digests
 * &#x2713; ChIP/ Co-IP: wash & elute (see 9/13/10)
 * &#x2713; RT-PCR: p16 induction cDNA from 9/13/10

Midiprep check > Check with KpnI digests

RT-PCR > Use cDNA from "p16 time course" samples > Same PCR conditions as p16 (see 8/26/10) > Templates + Primers:
 * 1) p16 time course (1-16) + p16 7C (0.5 uL cDNA)
 * 2) p16 time course (1-16) + mCherry f1/r1 (1.0 uL 1:1000 cDNA dln.)
 * 3) p16 time course (1-16) + GAPDH 21A (0.5 uL 1:1000 cDNA dln.)

--> Aliquot MM's: 19.5 for 1-16, 19.0 for 17-32, 19.5 for 33-48 --> Add cDNA --> 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: No signal for p16. Re-make cDNA with 5 ug RNA. Non-specific mCherry, use less template next time.


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