User:Karmella Haynes/Notebook/Polycomb project/2010/04/29

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


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04/29/10

 * &#x2713; RT-PCR: 4-day dox induction of Pc-ATF lines, mCh primer optimization
 * &#x2713; Senescence assay: expand KAH126-3, -4 cell cultures to 10 cm plates (maintenance med.)
 * &#x2713; Minipreps: FlpE (4)

RT-PCR

> Reaction 1: optimization of mCherry primers --> cDNA templates
 * 1) KAH126-1 (1:1)
 * 2) KAH126-1 +dox (1:1)
 * 3) KAH126-1 (1:10,000)
 * 4) KAH126-1 +dox (1:10,000)
 * 5) FTRx (1:1)
 * 6) FTRx +dox (1:1)
 * 7) FTRx (1:10,000)
 * 8) FTRx +dox (1:10,000)

--> primers
 * 1) mCh f1/r1, 100 bp, #1-8
 * 2) mCh f2/r2, 100 bp, #9-16

--> 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/ 30 sec.] x35
 * 72°C/ 3 min.
 * 4°C/ ∞

--> Result: primer pair one shows expected results at 1:10,000 dilution --> Next PCR: full Pc-ATF sample set, plus gene target primers

Minipreps > Check with E, P, and E/P digests

15 μL --> 37°C/ ~15 min.

--> None look correct. Next time, do a "slow" transfection (heat shock, recovery).


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