Cell culture and transfection

Dear Mr. Chin, 

You can follow the following protocol to transfect plasmid DNA or sgRNA/mRNA into mESCs or HEK293 cells in 96-well plate:

1. Plate 10,000 cells in 100 µl culture medium in 96-well plate on Day 0
2. Prepare transfection mix for plasmid DNA (A) or sgRNA/mRNA (B), and transfect cells on Day 1

A: Dilute 100 ng plasmid DNA in 10 µl Opti-MEM (Themo-Fisher), dilute 0.25 µl Lipofectamine 2000 (Themo-Fisher) in 10 µl Opti-MEM. Combine DNA and Lipofectamine 2000 solution, keep the transfection mixture at room temperature for 5 minutes, and add the transfection mixture to pre-plated cells.

B: Dilute 50 ng sgRNA and/or 100 ng mRNA in 10 µl Opti-MEM (Themo-Fisher), dilute 0.25 µl Lipofectamine MessengerMAX (Themo-Fisher) in 10 µl Opti-MEM. Combine sgRNA/mRNA and Lipofectamine MessengerMAX solution, keep the transfection mixture at room temperature for 5 minutes, and add the transfection mixture to pre-plated cells.

3. Change medium to remove  the transfection mixture 24 hours after transfection.

Cheers

Li