Transient HEK293T Transfection
Note
If performing transient transfection for virus production: please use the updated Virus production in HEK293T protocol for lentivirus production via HEK293Ts, and the Plat-E Retroviral Production protocol for Plat-E retrovirus production.
If performing transient transfection for an experiment, continue to the protocol below.
Calculations
For all calculations you can refer to the most updated transfection template in the shared projects folder of the Galloway Lab sharepoint, example here.
To scale per well amounts according to plate size, divide by number of wells with the total amount per entire plate constant (e.g., 1 10cm-dish = entire 6-well plate = entire 96-well plate). For instance, each well in a 6-well plate would use \(\frac{1}{6}\) the per-plate DNA and knockout DMEM amounts.
For conditions with multiple wells, make 110% of the calculated amount to account for pipetting loss. Additionally, make 120% KO DMEM + PEI MM to ensure there is enough extra.
Knockout (KO) DMEM + PEI Master Mix (MM) Calculations
Overall parameters:
Parameter |
Value |
|---|---|
Titrated PEI ratio |
Specific for each PEI batch (e.g., 5 ug PEI : 1 ug DNA). The PEI concentration itself is 1mg/mL. |
DNA/plasmid/well |
10.8 ug/plate |
KO DMEM/plasmid/well |
1.33 mL/plate |
Example MM calculation (make 120% for extra):
Value |
Example in a 96-well plate |
|---|---|
Number plasmids/well |
2 plasmids (example) |
Total wells |
50 wells (example) |
Titrated PEI ratio |
5 ug PEI : 1 ug DNA (example) |
KO DMEM/well |
(1.33 mL/plate/plasmid) / (96 wells/plate) * (2 plasmids) = 27.7 uL/well |
PEI/well |
(10.8 ug DNA/plate) / (96 wells/plate) * (5ug PEI / 1ug DNA) / (1 ug/uL PEI) = 0.56 uL/well |
Total KO DMEM (120%) |
(27.7 uL/well) * (50 wells) * 1.2 = 1.663 mL |
Total PEI (120%) |
(0.56 uL PEI/well) * (50 wells) * 1.2 = 33.75 uL |
Example condition mix calculation (make 110% for extra):
Value |
Example |
|---|---|
Plasmid concentration |
100 ng/uL (example) |
Wells/condition |
3 wells (example) |
Plasmid volume/condition (110%) |
(10.8 ug/plate) / (96 wells/plate) / (0.100 ug/uL) * (3 wells) * 1.1 = 3.71 uL |
Master mix/condition (110%) |
(~ 27.7 uL/well KO DMEM) * (3 wells) * 1.1 = 91.41 uL |
Protocol
Seed healthy 293T cells 1 day prior to transfection. Cells must be ~80% confluent at time of transfection for highest efficiency. Recommended cell counts are:
Cell Type |
Well Size |
# Cells/Well |
|---|---|---|
HEK293T |
96-well |
25-40K |
HEK293T |
10-cm |
7.5M |
Plat-E |
6-well |
800K |
Make a mastermix (MM) of PEI and Knockout DMEM according to the calculations above.
Important
Ensure that you add PEI to KO DMEM, not the other way around! Mix master mix well, then let sit for minimum 10 minutes.
For each condition, combine the DNA and the PEI+KO DMEM MM according to the calculations, then mix and wait 10-15 minutes. These are the “condition mixes.”
Gently add the calculated amount of condition mix to each desired well on TOP of the existing growth media using the 100% NOT the 110% volumes. For larger well (anything above 24-well), add the condition mix dropwise and evenly around the plate, rocking the plate back and forth, side to side to mix.
After 18-24 hours (1 day post transfection, dpt), aspirate and replace with fresh media (e.g., DMEM + 10% FBS for HEK293T). Typically, any small-molecule inducers are added at this step (e.g., doxycycline).
It is standard to image and flow cells at 2 dpt (1 day after fresh media change). If small-molecule inducers were added at 1 dpt, it is common to flow at 3 dpt to allow the expression levels to reach steady state.