User:Karmella Haynes/Notebook/Polycomb project/2011/04/15

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04/15/11

 * &#x2713; qRT-PCR: Pc-TF -/+ dox plates 23, 24
 * &#x2713; RNA prep/ cDNA: Try TRIzol/ RNeasy combo protocol; Sample #16 from HEK luc transfections (2-day/ mock)

qRT-PCR

> Plate 23 --> 750 nM Primers (& cDNA dilution)
 * 1) mCh1 (1:1000)
 * 2) GAPDH 21A (1:1000)
 * 3) ACTC1 35A (1:10)
 * 4) ATOH1 46D "
 * 5) CASP10 41A "
 * 6) CDKN2A 7C "
 * 7) EOMES 42A "
 * 8) GRHL2 37C "

> Templates, use 2 μL
 * 1) KAH157-1, 4/08/11 A
 * 2) KAH157-1 +dox, 4/08/11 A
 * 3) KAH157-1, 4/08/11 B
 * 4) KAH157-1 +dox, 4/08/11 B

> Plate 24 --> 750 nM Primers (& cDNA dilution)
 * 1) MMP12 31B (1:10)
 * 2) NPPA 40A "
 * 3) OTX2 43D "
 * 4) SST 45C "
 * 5) THPO 33A "
 * 6) TNF 34A "

> Templates, use 2 μL
 * 1) KAH157-1, 4/08/11 A
 * 2) KAH157-1 +dox, 4/08/11 A
 * 3) KAH157-1, 4/08/11 B
 * 4) KAH157-1 +dox, 4/08/11 B

--> Aliquot 39.0 primer mix into 1st well of each 3x set --> Add 6.0 (2.0 x3) DNA to 39.0 primer mix --> Aliquot 15.0 rxn mix to other 2 wells in each 3x set

Bio-Rad CFX96 qPCR (Kirschner lab) --> Use Bio-Rad 96-well low profile plate MLL-9601 + Microseal "B" film
 * 95°C/ 5 min.
 * [95°C/ 15 sec, 58°C/ 15 sec, 72°C/ 15 sec] x45
 * Melt curve range 58°C -> 95°C/ 0.5°C per step

RNA prep/ cDNA

> Sample "#16 2-day/mock"; frozen in TRIzol (-80°C) > Use TRIzol protocol up to chloroform extraction > Apply aqueous phase to RNeasy column (suggestion from Mara) and follow rest of RNeasy protocol > RNA prep: A260 = 1.393, 260/280 = 1.83, 55.7 ng/μL > Yield not very high, but peak looks good, decent quality RNA > cDNA synthesis: Superscript III kit; use 8 μL RNA (max) > Will use this cDNA to test luc primers when they arrive


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