There are many reasons your transformation may be less efficient than expected.  If the solutions in the table below do not help, please contact Invitrogen Technical Support

 

Possible Cause Solution
Impurities in the DNA For chemically competent cells, remove phenol, proteins, detergents, and ethanol from the DNA solution. For electrocompetent cells, ethanol precipitate ligations to clean up plasmid DNA, since salt and buffers severely inhibit electroporation and increase the risk of arcing. In addition, dissolve the DNA in sterile water or 0.5X TE (5 mM Tris-HCl, 0.5 mM EDTA).
Excess DNA or volumeAdd 1 to 10 ng of DNA in no more than a 5-µl volume per 100 µl of chemically competent cells. For Subcloning Efficiency™ cells, use 1 to 3 µl per 50 µl of competent cells. For ElectroMAX™ cells, add 1 µl (1 to 50 ng) to 20 to 25 µl of cells.
Inhibition of transformation by ligationFor One Shot®, MAX Efficiency®, Library Efficiency® and Subcloning Efficiency™ cells, dilute the ligation reaction mix 5 times with 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA before adding to competent cells.
Poor expression of antibiotic resistanceStore at -80°C. Invitrogen electrocompetent and chemically competent cells are stable for up to 2 years. Do not store cells in liquid nitrogen. Minimize the number of freeze-thaw cycles. Aliquot and refreeze any unused cells. Note, however, it will lower transformation efficiencies
Improper handling of competent cellsThaw competent cells on ice, and use cells immediately upon thawing. Do not vortex.
Improper heat-shock procedure for chemically competent cellsFor One Shot®, MAX Efficiency®, and Library Efficiency® cells, incubate cells at 42°C for 45 sec. without shaking. These conditions are optimized for round-bottom polypropylene tubes (17 to 100 mm) and 100 µl of cells. For Subcloning Efficiency™ cells, incubate cells at 37°C for 20 sec. using 1.5-ml microcentrifuge tubes and 50 µl of cells. For Stbl2™ cells, heat at 42°C for 25 sec. instead of 45 sec.
  • If there is a change in the tubes or volume of cells, the heat shock conditions must be optimized.
Improper electroporationUse devices that apply 16 kV/cm and the appropriate conditions for each electrocompetent strain
Slow or no growth of cellsIf cells are being grown at 30°C instead of 37°C, incubate for at least 90 min. during recovery and incubate the transformed colonies longer.
Overgrowth (little or no selection)Be certain that the correct antibiotic and correct concentration of antibiotic is used. See recommended usage in product manuals. Use fresh antibiotics—make sure the drug is not expired.