User:Karmella Haynes/Notebook/Polycomb project/2013/06/02

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06/02/13

  • Transfection: KAH126 for flow cytometry on Monday



Lipo transfection
> Cells were 100% confluent
> Re-plate 1:2 dilution prior to transfection in p/s-free medium


Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH126 2 μg 9.0 μL 4 μL 500 μL
2 KAH126 1 μg 4.5 μL " "
3 KAH126 0.5 μg 2.3 μL " "
4 KAH126 0.2 μg 0.9 μL " "
5 KAH126 0.1 μg 0.5 μL " "
6 none none --- none none

> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min.
> Add DNA to 250 μL Opti-MEM
> Add DNA mix to Lipo mix --> R.T./ 20 min.
> Remove 500 μL medium from each well; Add 500 μL complexes to each well (4 ml med. each)
> Add 1 μg/μL dox
> Grow cells at 37°C overnight


Next day
> When harvesting cells for flow cytometry, use 1/4 of cells to seed new plate (for outgrowth)
> Might run cells again to see when RFP reaches steady state



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