CaPO4 Transfection into 293T cell
(Also tried to transfect into CHO cell based lec8 glycosylation machinery deficient cell)
1. Prep solution
1. 2x HBS (Hepes buffered saline) ? 20ml
Hepes (acid): 0.2 g (Sigma H3375)
NaCl: 0.32g
Na2HPO4 (1M): 36ul
NaOH (5M or 1M) for pH adjustment
? Make fresh buffer as possible as you can. Adjust pH 7.12 (Crucial) by adding NaOH.
Bring up volume to 20ml and filter solution with steril filter.
2. 2M CaCl2 tissue culture grade from Sigma C7902
3. Chloroquine (10mM) sigma C6628, 51.6mg/10ml in ddH2O. Keep in dark and store at -20C
2. Procedures
1. Make appropriate cell confluency ~70 ~80 % (For example T-75)
2. Mix vector DNA with sterile water ( 15ug DNA in 480 ul total volume)
3. add 66ul of 2M CaCl2 into the DNA solution, mix well, and incubate on ice for 5min
4. add 555 ul of 2x HBS solution drop by drop, while simultaneously mixing the solution by vigorous agitation (like vortex)
5. Mix again and rest on ice for 20min
6. Change media with fresh one (12ml for 1 T-75 plate) during waiting for step 5.
7. Add the mixture into cell culture media drop by drop , mix gently
8. Add 50ul of Chloroquine (10mM) to media
9. Incubate the culture for 5-8 hours at 37C, 5% CO2 and then change media with fresh one.
10. Harvest media for every 24hrs or 12hrs.
