Mammalian Cell Transfection Kit Protocol

Note: Work with kit reagents at room temperature in a tissue culture hood or other sterile environment.

Plate cells 24 hours before transfection. Usual plating density is 1x106 cells/100 mm dish/ 10 mL complete media

Note: If transfecting a suspension culture, suspension cell concentration should be 5x106/mL. Suspension cells grown in RPMI may be difficult to transfect with this kit. It is recommended to use a DMEM for suspension transfection in this case.
Feed cells fresh, complete media 3 hours before transfection.
All DNA used should be phenol, phenol/chloroform/isoamyl alcohol extracted ethanol precipitated and dissolved in sterile UltraPure water or a tris/EDTA solution.

For each 100 mm plate of cells to be transfected, 1 mL of calcium phosphate precipitate is needed.

Note:If transfecting a suspension culture, suspension cell concentration should be 5x10⁶/mL. Suspension cells grown in RPMI may be difficult to transfect with this kit. It is recommended to use a DMEM for suspension transfection in this case.
Prepare 1X HBS fresh for each experiment. 0.5 mL of 1X HBS is neeed for each 100 mm plate.
Formula for 1X HBS is as follows:
- Add 0.88 mL sterile UltraPure water to tube.
- Add 0.1 mL 10X HBS and mix well.
- Add 15 µL NaOH solution and mix well.
  
  Note: The pH will be correct and need not be checked).

Set up two sterile polypropylene tubes for each DNA to be precipitated, label tubes #1 and #2 along with the DNA to be used.
Add to tube #1: 0.5 mL of 1X HBS and 10 µL phosphate solution.
Add to tube #2: 0.43 mL of UltraPure water minus volume of DNA. Total DNA should equal 20 µg.

Note: If genomic DNA is being used, the total DNA should equal 30 µg. Genomic DNA will replace carrier and plasmid DNAs.
Gently mix the DNA into the water.
Add 60 µL of calcium solution and mix gently.

Place a sterile 1 mL pipette into tube #1 and gently bubble air through the solution so that it is slowly mixing.
Draw the contents of tube #2 up into an appropriately sized sterile pipette. Add slowly, dropwise, to the gently bubbling and mixing solution in tube #1. As the two solutions mix, they will appear milky and then form a white precipitate. Continue to bubble and add slowly until the entire contents of tube #2 have been added.
Allow the suspension to sit at room temperature for 20 minutes before adding to the cells.

Mix the precipitate well by pipeting or vortexing, making sure that any large clumps that may have formed on the bottom of the tube are broken up and that the precipitate is evenly resuspended.
Add 1 mL of suspension to a 100 mm plate containing 10 mL of complete medium. The suspension must be added slowly, dropwise, while gently rocking the medium in the plate.
Return the plates to the incubator and leave the precipitate on for 12–24 hours.

Remove the medium containing the precipitate and add fresh complete medium leaving this medium on for 24 hours.
Remove the medium and add the appropriate selection medium to select stable colonies or add complete medium for transient expression incubation.

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