User:Karmella Haynes/Notebook/Polycomb project/2011/03/23

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


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03/23/11

 * &#x2713; qRT-PCR: test new primers

qRT-PCR primer test --> 750 nM Primers (& cDNA dilution); 30 μL 10 μM primer mix + 370 μLdH2O
 * 1) CASP10 41A
 * 2) CASP10 41D
 * 3) EOMES 42A
 * 4) EOMES 42D
 * 5) OTX2 43D
 * 6) SST 45C
 * 7) SST 45D
 * 8) ATOH1 46A
 * 9) ATOH1 46D

--> Templates, use 2 μL
 * 1) KAH126-1 +dox, 3/10/11, 1:10
 * 2) KAH126-1 +dox, 3/10/11, 1:100
 * 3) no cDNA

--> Aliquot 36.0 template mix into 1st well of each 3x set --> Add 9.0 (3.0 x3) primers to 36.0 template 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

--> Results (Ave C(t), 1:10 cDNA)
 * 1) CASP10 41A: 32.75 (use this primer)
 * 2) CASP10 41D: 33.06
 * 3) EOMES 42A: 37.63 (use this primer)
 * 4) EOMES 42D: 42.23
 * 5) OTX2 43D: 33.63 (use this primer)
 * 6) SST 45C: 34.93 (use this primer)
 * 7) SST 45D: 36.85
 * 8) ATOH1 46A: 36.99
 * 9) ATOH1 46D: 33.44 (use this primer)


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