CALCIUM PHOSPHATE TRANSFECTION

Reagents:

1. 2X HEBS (HEPES Buffered Saline)

· NaCl - 8.0g

· KCl - 0.37g

· Na₂HPO₄ - 0.099g

or Na₂HPO₄.2H₂O - 0.125g

or Na₂HPO₄.7H₂O - 0.188g

· Glucose - 1.0g

· HEPES - 5.0g

· Dissolve content in 450ml distilled water and set pH 7.2 for plasmid transfection and pH 7.05 for genomic DNA transfection.

· Make up total volume of solution 500ml by distilled water.

2. 2.5M CaCl₂ in 10mM HEPES

· HEPES - 0.238g

· CaCl₂ - 27.75g

· Dissolve content in 90ml distilled water and set pH 7.2 for plasmid transfection.

· Make total volume 100ml by distilled water.

Protocol (A): (For 6 well plates)

1. Plate cells (3.5X10⁵ cells in each well) the night before to attain 50-60% confluency at the day of transfection in a 6 well plate.

2. One hour prior to transfection, change the medium. Volume should be 2ml in each well.

3. Prepare two sets of 15ml tubes: (for total 6 wells)

· Solution A:- 900µl 2X HEBS

· Solution B: - 12µg DNA in 810 µl distilled water + 90µl 2.5M CaCl₂.

(Mix DNA and CaCl₂ by drawing up and down by a 1ml pipet)

4. Add Solution B drop wise to Solution A and gently mix by drawing up and down.

5. Add this mixture (300µl in one well) directly to the cells by sprinkle to the entire surface of medium. Do this within 1-2 minute after adding DNA and CaCl₂ to the 2X HEBS.

6. Swirl the plate gently to mix.

7. Incubate for 10 hours.

8. After incubation replace the CaPO₄ containing medium with normal medium and incubate for 24-48 hours.

Protocol (B): (For 10cm plate)

1. Plate cells (2.0X10⁶ cells) the night before to give 50-60% confluence at the day of transfection in a 10cm plate.

2. One hour prior to transfection, change the medium. Volume should be 10ml.

3. Prepare two sets of 15ml tubes:

· 500µl 2X HEBS

· 10µg DNA in 450 µl distilled water + 50µl 2.5M CaCl₂.

4. Mix DNA and CaCl₂ by drawing up and down by a 1ml pipet.

5. Add DNA solution drop wise to 2X HEBS and gently mix by drawing up and down.

6. Add this mixture (1000µl) directly to the cells by sprinkle to the entire surface of medium. Do this within 1-2 minute after adding DNA and CaCl₂ to the 2X HEBS.

7. Swirl the plate gently to mix.

8. Incubate for 10 hours.

9. After incubation replace the CaPO₄ containing medium with normal medium and incubate for 24-48 hours.