TS Cell Lipofectamine

TS Cell Lipofectamine  DSM
February 11, 2010
  • Start
    Transient Transfection of TSC Using Lipofectamine
1. Day 0 Plate 5x105 TSC/3 ml in each C6 well (to be subconfluent the next day)
2. Day 1 In sterile tube:
1 µg specific gene plasmid
0.2 µg reporter plasmid
212 µl RPMI 1640/PLUS Mix		 RPMI 1640/PLUS Mix: For each C6 well add:
200 µl RPMI 1640 - no additions
12 µl PLUS reagent
(plus slight extra)		 RPMI 1640/Lipofectamine Mix: For each C6 well add:
190 µl RPMI 1640 - no additions
10µl Lipofectamine
(plus slight extra)
3.   15 min at room temperature
4.   Add 200 µl RPMI 1640/Lipofectamine Mix to each tube containing DNA/PLUS
5.   15 min at room temperature
6.   Wash TS cells in PBS
7.   Add 800 µL RPMI 1640 to each TSC/C6 well
8.   Add DNA/PLUS/Lipofectamine solution to TSC/C6 well
9.   Incubate at 37°C/5% CO2 for 3 h
10.   Add 2 mL FCM with 50 ng/mL FGF4 and 2 µg/mL heparin
11.   Incubate at 37°C/5% CO2 for 16 h
12. Day 2 Change the medium to 70% FCM with F4H
13.   Incubate at 37°C/5% CO2 for 24 h
14. Day 3-? Assay for reporter gene activity