How do you do a reverse transfection?

In reverse transfection protocols, cells are added directly to a plate containing the transfection reagent:DNA mixture and then assayed on the second day. This approach reduces the experimental time by a day compared to standard transfection protocols.

How long does Lipofectamine take to work?

Depending on the construct used, transiently expressed transgene can generally be detected for 1 to 7 days, but transiently transfected cells are typically harvested 24 to 96 hours post-transfection.

Is reverse transfection more efficient?

High efficiency of the reverse transfection decreases the amount of nucleic acid used. Unlike forward transfection, the transfection reagent can remain in contact with the cells for 24-72 hours.

How to transfect HEK293 cells using Lipofectamine 3000?

I am currently having problems with my transfections and request help. I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% confluency. Also, I used 2500 ng of DNA per well, 5µl of P3000 per DNA, 5µl of Lipofectamine 3000, and DMEM with P/s + L-glu instead of OMEM.

How is Lipofectamine used as a DNA transfection reagent?

Lipofectamine®2000 DNA Transfection Reagent Protocol Transfect cells according to the following chart. Volumes are given on a per-well basis. Each reaction mix is sufficient for triplicate (96-well), duplicate (24-well), and single well (6-well) transfections, and accounts for pipetting variations.

How are 293f and 293T cell lines related?

The 293F cell line is a fast-growing variant of the 293 cell line originally obtained from Robert Horlick at Pharmacopeia while the 293T cell line is a variant of 293 cells that harbors the SV40 large T antigen which can bind to SV40 enhancers of expression vectors to increase protein production.

How to transfect NIH3T3 cells with SWIS 3T3?

1. Pre-warm 50ml of Optimum (stored in cold room at 4°C) ~10min. 2. Add ~2g of DNA to Eppendorf tube in the hood. µ 3. Add 100l of Optimum to the Eppendorf tube to dilute the DNA and mix by µ tapping. 4. Add 6l of Lipofectamine2000 (from Biostores) and 100µ µl of Optimum to another Eppendorf Tube. 5. Combine #3 (DNA) and #4 (lipofectamin). 6.