how to test the efficiency of chemically competent cells - (May/04/2008 )
I just made some DH5alpha chemically competent cells (CaCl2 method). Is there a way that I can test them to see how efficient they are?
Determine the transformation efficiency of your bacteria using a commercial supercoiled plasmid. Briefly, add a predetermined amount of supercoiled DNA (any size) into predetermined volume of competent cell. Transform and plate predetermined vol of transformation rxn onto agar (dilution might be required). After that, do calculation on transformation using the method described here (http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/Cloning_and_Expression/Key_Resources/Transformation_Efficiency.html). Or you can key in info onto this webtool (http://www.sciencegateway.org/tools/transform.htm) to determine the transformation efficiency of your bacteria. If not mistaken, it's 10^5 to 10^6 cfu/ug DNA is the best for DIY competent cell (Some protocol might be higher).
Get a known concentration DNA plasmid sample -- we use the NEB pUC19 at 1 ug/ul. Do serial dilutions to 10 pg/ul. You may want to add BSA to reduce DNA sticking to the tube walls. Transform bacteria (typically 50 ul), grow up for 1 hour in non-selective medium and plate out on selective medium (amp in this case). If you plate only a portion of the cells, this will affect your calculation. Count colonies and calculate the number of colonies per microgram of DNA used for transformation (these are the somewhat random units this is usually reported in). For good chemically competent cells you should get 10^8 to 10^9. CaCl2 will only get you 10^6 or so. Electroporation will hit 10^10 or higher.