Protocols

Calcium phosphate transfection protocol

This is our lab’s calcium phosphate transfection protocol for HEK293 (293HEK) cells in 15 cm dishes: seeding density, CaPO4 precipitation, overnight incubation, and media change. The 1× HEPES buffered saline (HBS) recipe and DNA scaling table are included below.

Procedure

  1. Grow enough 293HEK cells in T150 flasks so that you have one T150 for two 15 cm dishes. 293 cells are normally grown in 10% FCS in DMEM (with Pen/Strep and Glu) but will also grow well in 10% FCS DMEM.
  2. On the morning of the day of transfection, trypsinise the cells and seed 1 T150 into two 15 cm dishes in 24 ml of 10% FCS/DMEM. Transfection must be performed in DMEM not EMEM. Do this as early as possible to allow the cells to stick down and spread.
  3. At about 3 pm, remove the 1× HBS and 2.5 M CaCl2 and warm up to room temperature.
  4. At about 4 pm set up the transfection as below. Set up a single 1.5 ml Eppendorf for each 15 cm dish.
    1. Add 1.3 ml of 1× HBS.
    2. Add 77 µg of plasmid and mix well by pipetting up and down.
    3. Add 38.5 µl of 2.5 M CaCl2 and mix immediately by rapidly pipetting up and down.
    4. Add another 38.5 µl of CaCl2 and mix immediately by rapidly pipetting up and down.
    5. Incubate at room temperature for 40 minutes.
    6. Add dropwise to each dish. Rock back and forth so you spread the transfection mix evenly.
  5. The next day you should see a very fine precipitate, particularly in areas where there are no cells. You will need to use the highest magnification in TCB to see this clearly. It will almost look like a bacterial infection.
  6. Remove the media and replace with cold fresh media (normally 10% FCS EMEM but DMEM will also do).
  7. Expression will only be good 48 hours after transfection.

1× HEPES buffered saline (1× HBS)

Table of DNA amounts for different culture vessel surface areas: format, relative surface area, media volumes, 1× HBS, 2.5 M CaCl₂, total DNA, and GFP plasmid ranges
Table of DNA amounts for different surface areas (35 mm dish = 1.0× reference).

About the author: This page was written by Dr Mark Bond from The Bond Lab at the University of Bristol. These notes reflect the methodology used in our cardiovascular and cell-signalling research. Questions about these methods: contact us or email mark.bond@bristol.ac.uk ORCID.