User:Karmella Haynes/Notebook/Polycomb project/2009/12/07

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12/07/09

 * &#x2713; Transfection: transfection and neo-induction of K-stop constructs

Transfection > Test of Neo-inducible translation

> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min. > Add 6x vol DNA --> R.T./ 20 min. > Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C > After 4-5 hours, refresh medium and add varying amounts of neomycin (G418); make serial dilutions (1:2) --> Initial dilution = 400 μg/ 100 μL (8 μL 50 mg/mL stock in 92 μL Opti-MEM) --> 4x (= 400 μL) = 32 μL 50 mg/mL stock in 368 μL Opti-MEM --> Final vol. of each dilution is 50 μL (4x = 200 μL)

> Assay RFP after 18-24 hours


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