Lipofection of SW1353 chondrosarcoma cells

Dominik Haudenschild at SCORE - August 2008

Usually I seed about 4-5x10⁵ SW1353 cells/well in 6 well plate, so that next day it will be around 95% confluence.

A few hours before transfection, change the medium to fresh medium or optimem, but no antibiotics added in this step.

Culture the SW 1353 cells with 10% FBS, somebody used 10% CS, but the cells were not happy.

For Lipofection,

1. add 10 ul lipofectamine 2000 into 250 ul optimem, gently mix by pipetting up and down a few time gently. Mix the lipofectamine by tabbing on the tube a few time before pipetting the reagent from the lipofectamine tube, it will even the concetration.

2. in another tube, add 4 ug of plasmid into the 250 ul optimem, mix by pipeting up and down a few time

3. add tube 2 into tube1, then pipetting up and down for a few times. then leave on RT for 25 min

4. add the mixture from step 3 drop wise into the well, mix by moving the plate up and down a few times, then left and right a few times.

5. next morning, change the medium into the normal 10% FBS/DMEM

6. you can either do experiment at 24 hours or 48 hours after transfection, we usually do after 48 hours.

7. cells lysed in 50ul sample buffer, load 15ul per well.