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Protocol

CaPO4-Transfection

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CaPO4-Transfection For 100 mm plate. Plate cells the day before at approximately 60-70% confluent. 1. Remove old media from plates and add 10 mls fresh media. 2. Add 1/2 ml 2x CaCl2 to 15 ml tube. 3. Add 20-30 ug DNA to each tube Optimal amount of DNA will vary with each DNA prep. DNA should be very pure. Use carrier DNA if specific DNA is limiting Ex: 20 ug calf thymus DNA (optimized to 25 ug) 5 ug 5 SV2 CAT DNA 4. Add 1/2 ml 2x BBS to each tube dropwise let each drop slowly slide down tube into CaCl2 - DNA solution 5. Tap gently and let stand at R.T. 5-1011 6. Add 1 ml mixture to each plate of cells and put cells at 350C, 3.5% C02 - O.N. 7. Next day should have very fine ppt. covering bottom of plate 8. Wash cells 3x with serum free media for each wash leave media on cells for 1-511 before replacing 9. Add 10 mls fresh media with serum and put at 370C, 6% C02 until ready to harvest. Normally 48 hrs after starting transfection 20x CaCl2 2.5 M CaCl2 in H20 18.3 g CaCl2 gs-- 50 mls 2x BBS - for 1 liter 50 MM BES (NW 213) Sigma cat. #B-9879 10.65 g 280 mM NaCl 16.464 g 1.5 MM Na2HP04 213 mg pH to 6.89 with NAOH
Ca PO4, Transfection (106 mm plates) 1. Remove old media from plates and add 10 mls fresh 2. Add 1/2 ml 2 X CaCl2 to 15 ml tube (can double reactions if desired) 3. Add 20 - 30 ug DNA to each tube -optimal amount of DNA will vary with @ DNA prep. -DNA should be very pure -can use carrier DNA if specific DNA is limiting ex: 20 ug calf thymus DNA (optimized to 25 ug) 5 ug SV2 CAT DNA 4. Add 1/2 ml 2 X BBS to each tube dropwise -let each drop slowly down tube into CaCl2-DNA solution 5. Tap gently & let stand at R.T. 5-101 6. Add 1 ml mixtures to each plate of cells and put cells at 350C, 3.5% C02 - O.N. 7. Next day should have very fine pcpt. coverning bottom of plate 8. Wash cells 3 X w/serum free media -for each wash leave media on cells for 1-51 before replacing 9. Add 10 mls fresh media w/serum and put at 370, 6% C02 until ready to harvest 20 X CaCl. 2. 5 M CaCl2 in H20 18.3 g CaCl2 qs=50 ml " X BBS 50 mM BES (KW 213) sigma Cat. #B-9879 10.65g 280 mM NACL 16.464g 1.5 nl4 Na2HP04 213mg pH to 6.89 w/NaOH qs=l liter 10. Harvest the cells 48 hours after transfecting them. Aspirate media, and wash 2 times with PBS. Add 1 ml cold TEN buf f er and let set for 5 minutes. Remove cells with a rubber policeman or cell scraper, and transfer to Eppendorf tubes. Spin 5 min. in a microfuge. Aspirate the buffer, and add 150 ul (300 ul for 100 mm plates) cold 0.25 M TRIS, pH 7.8. Sonicate cells with 6-1 sec. bursts. Spin for 5 min. again in a microfuge. The size of the cell pellet should be smaller than after spinning the whole cells. Transfer the supernatant to Eppendorf tubes and store frozen at -200C. 11. CAT Assav Add .25 M TRIS to Eppendorf tubes so that the final volume of buffer plus cell extract will be 150 ul. Then add: 25 ul Acetyl Co-A (Lithium salt, from PL Biochemicals), 3.52 mg/ml 5 ul 14 C-Chloramphenicol (NEN) cell extract. Vortex, and incubate for 60 min. at 370 Stop the reaction by extracting 2 X with 1 ml ethyl acetate. Dry the extract, then resuspend in 30 ul ethyl acetate. Spot 10 ul onto TLC plates. Chromatograph in 200 ml of 95:5 chloroform: methanol (takes about 1.5-2 hrs.) Expose to X-ray film O.N. at room temperature. TEN Buffer .4 ml TRIS pH 7.5 (lM) .2 ml EDTA (.5M) 3 ml NaCl (5M) 92.5 ml H20
References: Chen & Okayama - Biotechniques 6:632 (1958) Chen & Okayama (1987) - MCB 7:2745

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