Calcium PhosphateTransfection Kit 

No more bubbling and slow dripcalcium phosphate transfections. Our NEW Calcium PhosphateTransfection Kit is easy to use and will allow you to performtransfections on standard 60 mm or 100 mm dishes as well as 96-wellplates:
 

Kit Components

1.0 M Calcium Chloride (15 ml)

2x Phosphate Buffer (60 ml)

50 mM HEPES, pH 7.05, 23 °
1.26 mM Na2HPO4 
140 mM NaCl

Storage: 2-8 °, stable up to one year

 

Product 

Qty 

Catalog# 

Price

Calcium Phosphate Transfection Kit 

1 kit

74989

$90.00

Dish Size 

Number of Transfections

100/60 mm 

120-240

96 well plate

31

General Information 

  1. We encourage investigators to modify the following protocols according to the characteristics of the cell line(s) being used.  
  2. All cell lines should be tested for glycerol or DMSO sensitivity prior to carrying out transfection.  
  3. Reference: Jorden, M., Schallhorn, A., and Wurm, F.M. 1996. Nucleic Acid Res: 24(4): 596.  

Additional Reagents (not supplied with kit) 

  1. Serum-free culture medium, sterile  
  2. Complete culture medium, sterile  
  3. Distilled water, sterile  
  4. Glycerol or DMSO, sterile (used only with tolerant cells). 

Suggested Glycerol/DMSO Tolerance Test 

  1. Prepare 5 ml of Shocking Buffer (10, 15 or 20% v/v DMSO in complete culture medium).  
  2. Aspirate medium from cells.  
  3. Add 2 ml (100 mm dish) of Shocking Buffer to cells.  
  4. Test for tolerance by examining cells at 30 second intervals for 3 minutes. Round cells indicate that the cell line is not Glycerol/DMSO tolerant.  
  5. If most cells survive during the incubation period, determine the optimal shocking time for that cell line.  

Transfection Protocol 
This may vary depending on the characterisitcs of the cell line used. We generally recommend the following plating and transfection conditions listed in tables 1 and 2.

Table 1 

Plate or Well Diameter

Cells per Plate
(cell line dependent) 

DNA / Calcium Phosphate Suspension

100 mm

5-25 x 105 

1000 µl

60 mm

2-10 x 105 

500 µl

6 mm (96 well format)

4-10 x 103 

20 µl
 

Table 2 

Transfection Reagents

Culture Plate
(60 or 100 mm) 

96 Well Plate
(6 mm) 

Plasmid DNA 

1-30* µg

10-500* ng

Water 

up to 375 µl

up to 15 µl

1.0 M CaCl2 

125 µl

5.0 µl

2x Phosphate Buffer 

500 µl

20 µl

* The amount of plasmid DNA is dependent upon the cell line chosen.

Recommended Transfection Procedure

Day 1:  

Plate cells in complete culture medium 50 - 60% confluency 16 - 24 hours prior to transfection. 

Day 2: 

Prewarm transfection mediums and Shocking Buffer (the percentage is predetermined from Glycerol/ DMSO shocking test) to 37 °. 

Standard Culture Plate (60 or 100 mm) 

96 Well Plate (6 mm)
  1. Aspirate old medium and feed cells with 4.5 mL (60 mm dish) or 9.0 mL (100 mm dish) of fresh complete medium 1 - 2 hours prior to transfection. 
  2. Add 1 - 30 µg of plasmid DNA to a sterile 2 mL microfuge tube. Dilute with sterile water to 375 µL. 
  3. Add 125 µL of 1.0 M CaCl2 to the tube. 
  4. Add 500 µL of 2x Phosphate buffer to the tube, and quickly vortex the sample for 1 - 2 seconds. 
  5. Immediately add the DNA/Calcium Phosphate suspension* dropwise to culture plate and gently swirl medium for efficient mixing. 
    *Amount of DNA/ Calcium Phosphate suspension recommended: 
    0.5 mL per 60 mm dish 
    1.0 mL per 100 mm dish 
  6. Incubate cells under standard growth conditions for 2- 6 hours.
    For Glycerol/ DMSO tolerant cell lines: Aspirate medium from cells. Add 1 mL (60 mm dish) or 2 mL (100 mm dish) of Shocking Buffer . Swirl plate to cover cells with buffer. Incubate at room temperature for 1 - 3 minutes (the length of incubation is predetermined from Glycerol/ DMSO tolerance test). Aspirate Shocking Buffer and wash cells with 5.0 mL serum-free medium. 
  7. Remove medium and wash cells once with 5.0 mL (60 mm dish) or 10.0 mL (100 mm dish) of serum free medium.  
  8. Add 5.0 mL (60 mm dish) or 10.0 mL (100 mm dish) complete culture medium and incubate cells at standard growth conditions (typically 37 °, and 5% CO2 ). 
  1. Add 100 µL of complete culture medium per well 1 - 2 hours prior to transfection.  
  2. In a sterile U bottom 96 well plate, dilute 10 - 500 ng of plasmid DNA with sterile water to 15 µL per well. 
  3. Add 5.0 µL of 1.0 M CaCl2 to each well. 
  4. Add 20 µL of 2x Phosphate buffer, tap the plate on the bench once or twice to collect solution to the bottom of each well. 
  5. Quickly mix DNA/Calcium Phosphate suspension for 3 - 5 seconds by either pipetting up and down for 3 - 5 times or by shaking at 600 - 1000 rpm for 5 seconds. 
  6. Immediately transfer 20 µL of suspension to each well containing cells in culture. Swirl plate gently for efficient mixing.
  7. Incubate the cells under standard growth conditions for 2- 6 hours.
    For Glycerol/ DMSO tolerant cell lines: Remove medium* from cells. Add 20 µL of Shocking Buffer to each well. Swirl plate to cover cells with buffer. Incubate at room temperature for 1 - 3 minutes (the length of incubation is predetermined from Glycerol/ DMSO tolerance test). Add 100 µL of culture medium to wells. 
  8. Remove medium* and wash cells once with 200 µL of serum free culture medium.  
  9. Add 100 µL of complete culture medium per well and incubate cells at standard growth conditions (typically 37 °, and 5% CO2 ). 

*Remove medium by inverting plate onto sterile absorbent paper. 

Day 3: 

The appropriate antibiotics can be added to select transfectants 24-48 hours after transfection. 

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