How do you transfect HEK 293 cells?

The protocol for a 24-well transfection reaction with HEK293 cells is here:

1. Plate 10,000-15,000 HEK293 cells per well in 0.5 ml of complete growth medium 12-24 hours prior to transfection.
2. Wash with 1xPBS and add 0.5 ml of fresh growth medium.

How much DNA does it take to transfect in a 6-well plate?

Typically, for experiments in 6-well plates, 150 000 to 250 000 cells are seeded per well 24 h prior to transfection. For other culture formats, refer to Table 1. For DNA/siRNA co-transfection experiments, we recommend using 2 µg DNA and 10 to 50 nM siRNA per well in a 6-well plate.

What is the difference between lipofectamine 2000 and 3000?

Lipofectamine 3000 reagent yields higher transfection efficiencies than Lipofectamine 2000 reagent when tested in a variety of cell lines.

How do you take Lipofectamine?

Mix Lipofectamine 2000 gently before use, then dilute 1 μl in 50 μl Opti- MEM I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.

Does Lipofectamine expire?

According to ThermoFisher, lipofectamine 3000 can be used until 2 years after the manufacturing date if stored properly (stored in a 4 degree fridge should be fine). The Optimem is sensitive to expiration. I would recommend using the new reagents for your experiment. Hope this helps!

Why is HEK293 used in transfection?

It contains the SV40 large T-antigen that allows for episomal replication of transfected plasmids containing the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of desired gene products.

What are HEK293 cells?

HEK293 are immortalized human embryonic kidney cells. Originally isolated in the 1970s by Alex Van der Eb, a Dutch biologist, it was postdoc Frank Graham who transformed the cell line with sheared adenovirus 5 (Ad5). The ‘293’ refers to the fact that it was Graham’s 293rd experiment.

Do I need to remove Lipofectamine?

Make DNA-Lipofectamine™ 3000 complexes in serum-free medium such as Opti-MEM™ Reduced Serum Medium and add directly to cells in culture medium, in the presence or absence of serum/antibiotic. 2. It is not necessary to remove complexes or change/add medium after transfection.