User:Karmella Haynes/Notebook/miR Trigger/2009/12/06

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

12/06/09

  • ✓ Transfection: KAH93, 94, 95/MV2 (RFP); KAH99, 100, 101/MV2 (CFP)

Transfections
> U2OS cells + Gal4DB-PEST-RFP constructs
> Use Lipofectamine, 6-well format

Wells Plasmid DNA Volume Lipo Opti-MEM (total)
1 KAH93/MV2 2 μg 4.4 μL 4 μL 500 μL
2 KAH94/MV2 " 4.8 μL " "
3 KAH95/MV2 " 8.7 μL " "
4 KAH99/MV2 " 15.2 μL " "
5 KAH100/MV2 " 10.4 μL " "
6 KAH101/MV2 " 7.5 μL " "
7 KAH36(control) " 2.5 μL " "
8 mock --- --- " "
9 untreated --- --- --- "

> 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.
~~Cells in 6-well plates too crowded, discard; do same-day plating/ transfection from back-up stocks~~
> Harvest cells, plate 5x105 cells/3.5 ml ab-free medium in 6-well plates > Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37°C
> Check RFP & CFP expression (microscopy) after 18-24 hours


Personal tools