User:Karmella Haynes/Notebook/Polycomb project/2010/09/07

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


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09/07/10

 * &#x2713; RT-PCR: Pc-ATF Nanostring set, mCherry; test HHEX primers on KAH129-4
 * &#x2713; Sequencing order: KAH160-170/KAH015(FUW) for Casey (trouble expressing some constructs; check via sequencing)
 * &#x2713; Order oligos: new gene subset (see 9/05/10)

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 (1:100 cDNA dln.)
 * 2) Ns. KAH129-4 -/+ dox + HHEX 28A
 * 3) Ns. KAH129-4 -/+ dox + HHEX 28B

--> Add 5.0 μL cDNA (1:1000) to 1-20 --> Add 0.5 μL cDNA (1:1) and 1.0 μL primers to 11-14 --> 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 products for mCherry (re-do with 2 uL template); HHEX gave no signal

Order oligos
 * IL4
 * MMP12
 * NPPA
 * THPO
 * TNF


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