Editing 293T Protein Production (section)

== Small Scale (6-well)<br>  ==

1. Use T75 cm2 or T225 cm2 cell culture flasks to grow the HEK 293T cells and split the cells every 2-3 days when cells are 100% confluent.<br> <br> 

2. Seed 2.5 x 10<sup>5</sup> HEK 293T cells per well in a 6-well plate and add 2 ml DMEM with 1X pen/strep and 10% (v/v) FBS, swirl the plate gently to ensure even cell dispersal in each well, and incubate overnight at 37 °C in a 5% CO2 humidified chamber.<br> <br> 

3. When HEK 293T cells reach 40% confluency, discard the media and add fresh 2 ml DMEM with 1X pen/strep and 10% (v/v) FBS to the wells. Perform transfection assays.<br> <br> 

4. Aliquot 90 μl serum-free DMEM into a sterile 1.5 ml microcentrifuge tube. Pipette 3 μl L2K into the serum-free DMEM and gently mix the tube (finger vortex). Incubate for 5 minutes at room temperature.<br> <br> 

5. Add 1 μg of MiniPrep purified plasmid DNA (DNA stock= 100 ng/μl) into DMEM-L2K mixture, finger vortex, and incubate for 15 minutes at room temperature.<br> <br> 

6. Pipette the transfection mixture dropwise onto HEK 293T cells, and swirl the 6-well plate gently to allow even distribution of the transfection mixture. Incubate the 6-well plate at 37 °C in a 5% CO2 humidified chamber.<br> <br> 

7. Add 1 ml fresh DMEM with 1X pen/strep and 10% (v/v) FBS to each well 24 hours post-transfection and incubate for another 48 hours (total 72 hours).<br> <br> 

8. Harvest 1 ml of supernatant from each well at three days post-transfection and microcentrifuge the samples at 16,000 g for 10 minutes at room temperature.&nbsp; Samples can be stored at 4 °C. The length of storage at 4 °C is protein dependent.<br>