User:Karmella Haynes/Notebook/Polycomb project/2010/03/02

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


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03/02/10

 * &#x2713; RNA prep/ cDNA: 130-2 (4 day dox) samples
 * &#x2713; RT-PCR (continue tomorrow)

RNA Preps/ cDNA synth.

> RNA Preps --> Use 1 mL TriZol/ well (6-well dishes) --> Resuspend RNA pellet in 10 μL THE RNA Storage Solution

> oligo(dT) Primer annealing

> cDNA synthesis mix --> 2 reactions total

--> Add 10 μL mix to each annealing rxn. --> 50°C/ 50 min., 80°C/ 5 min., ice --> Add 1.0 μL RNase H, 37°C/ 20 min. --> Store at -20°C

RT-PCR

> Reaction 1: Loading optimization --> 4-day dox induction set, plus EZH2 k.d. (8 day) and nt (8 day) --> Use GAPDH primers and 1:10,000 cDNA template dilutions --> Note: EZH2 kd (8 day) (dilute 3x)

--> 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/ ∞

> Reaction 2: gene activation assay

--> Primers/ cDNA dilution:
 * 1) MYT1 (1D)/ 1:1
 * 2) MYT1 (1G)/ 1:1
 * 3) p15INK (5)/ 1:1
 * 4) p16INK (7B)/ 1:1
 * 5) RUNX3 (8A)/ 1:1
 * 6) DUSP4 (15A)/ 1:1
 * 7) TGFB1 (10A)/ 1:100
 * 8) PLAU (12A)/ 1:100
 * 9) KRT17 (14A)/ 1:100
 * 10) LAMB3 (16A)/ 1:100
 * 11) PPPIR (17A)/ 1:100
 * 12) GAPDH (21A)/ 1:10,000

--> Aliquot 9.0 μL of each DNA mix to appro. wells --> Aliquot 11.0 μL of each primer mix to appro. wells --> 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/ ∞




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