Calcium Phosphate Transfection Kit

We recommend that no antibiotics are present during transfection.  The process of transfection can make the cells somewhat more porous to allow for efficient DNA entry.  During this time, antibiotics will also enter the cells more easily and the cells may show increased cell death.  Wait until about 24 hours after transfection to resume the use of preventative antibiotics and/or start the use of selective antibiotics.

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The size of the plasmid should be considered when selecting a transfection reagent with the best efficiency.  In general, larger sized plasmids should easily transfect with readily available transfection reagents, as along as the plasmid DNA is of high purity.

For many common cell lines, transfection reagent efficiency is very high and the protocols will not require any optimization.  For hard-to-transfect cells or those ultimately expressing a toxic protein, the protocol should be optimized for best transfection efficiency.  Taking time to optimize will give you more transfected cells with each procedure, which can mean more protein expressed and results that are reproducible.

Yes, we recommend cells are at a low passage when being  used for any application, including transfection.  The reason why depends on what type of cells they are.  Primary cells will undergo a finite number of divisions, and as they get closer to senesence they divide more slowly - both affecting their ability to take up DNA (transient transfection), and minimizing their abillity to incorporate the DNA into the genome (stable selection).Cultured common cell lines are often immortalized, and generally continue to aquire mutations, leading to a heterogenous population that may perform differently from cells of lower passage number - leading to results that are not reproducible.

The calcium phosphate transfection method is performed differently than other methods, and so it can be optimized beyond the general recommendations.  An important part of the protocol is generation of a fine precipitate.  Be sure that the bubbling in tube B is consistant and with good force, while the solution from tube A is added very slowly, dropwise.  Once the precipitates are added to the cell culture, be sure they are evenly distributed on the plate so all cells may be transfected.The pH of the HeBS is critical to precipitate formation.  Prolonged storage of the solution may cause the pH to shift away from the optimal 7.05 - 7.12.

Transfection efficiency is a measure of how many cells take up the DNA during the transfection process.  Many transfection reagents can achieve a transfection efficiency of >90% in common cell lines.  Other cell lines are hard to transfect, and require special reagents and/or techniques to achieve even a small population of transfected cells.

For most transfections, cells should be >70% confluency the day of transfection, and growing in mid-log phase.  Some transfection reagents are now designed to work with cells at low density, when required.

The precipitate is normal to see - this is the calcium/DNA precipitate the cell will internalize during the transfection process.  If bubbled correctly, the precipitates will be very fine and regular sized, then evenly distributed over the cells.

There are many guides that help you select a transfection reagent.  In general, consider:The type of cell(s) you will transfectThe type of nucleic acid or protein you will introduce to the cellThe composition of your cell culture mediumThe need for stable or transient transfectionThe equipment you have availableThe other factors important to you - cost, protocol flexibility, ease of use, etc.