Recently, I have had problems with my transfection efficiency after I freeze and thaw my HBS2x. I use HEK293 cells and transfect them with calcium phosphate. My HBS2x recipe is: 50 mM HEPES, 280 mM NaCl, 1.5 Na2HPO4, pH 7, and I use 2M CaCl2. I mix the H2O, CaCl2 and DNA and slowly add this mix drop by drop in my HBS2x while mixing it, then I wait 20 min at room temperature and add this mix to the cells drop by drop while swirling gently the plate. I have done pH curves and different CaCl2 concentration as well as DNA concentrations so I have a 50% transfection efficiency using these conditions but only if the HBS2x is fresh, after i freeze it (-20ºC) and thaw it and repeating the same protocol the efficiency is 5%, any suggestions?
It's not very good to begin with. You should get 95% of HEKs with an optimal protocol, not 50%.
The further drop is most likely due to a subtle pH change. Phosphate buffers do not freeze well. You should basically never do that. The acid and the base precipitate at different rates due to freezing, and go back in solution at different rates, or not at all. Try to measure the pH if you have a sensitive meter, and look for precipitates with a good microscope. CaPO4 transfection is ridiculously sensitive to that.
We routinely transfect 293T cells using calcium phosphate method as described below.
2x Hank’s buffered saline (HBS) (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.1). (aliquots stored at -20°C)
2.5 M CaCl2
Day 1. Prepare a DNA precipitation mixture:
For 500 µl of transfection mixture (sufficient for each well in a 6 well plate) combine:
250 µl of 2x HBS
plasmid DNA
Sterile H2O (volume to 475 µl, can be added before plasmid DNA) and mix gently.
25µl of 2.5M CaCl2 dropwise while mixing the solution (CaCl2 must be added last to the precipitation mixture)
Leave the transfection mixtures at room temperature for 20 min with occasional mixing.
Remove medium from the cells. Immediately add 500µl of DNA precipitate and leave at room temperature for 30 min with occasional gentle tilting of plate. At this stage, precipitates can be seen under the microscope.
Add 4.5 ml of complete DMEM to each well and incubate plates at 37°C/5%CO2 or 10%CO2, as appropriate, for 12-14 hours.
Day 2. Transfected cells are subjected to osmotic shock: Remove medium from transfected cells and add slowly 0.5 ml of 10% glycerol in serum free DMEM at the edge of the each well, tilting the plate to ensure complete coverage.
Leave plate at room temperature for 4 min with gentle tilting.
Gently wash cells twice with serum free DMEM. (We find that osmotic shock is not necessary for 293T cells)
Add 3 ml of complete DMEM and culture as required.
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