Competent Cells
 | Transformation efficiency. Genetic markers. Packaging formats that fit your workflow. These are the most important considerations when choosing competent cells for your cloning experiment. Each of these factors will directly impact the time and effort required for your project, as well as its success. That’s why Invitrogen offers you a number of cell types and formats. The factors and the attributes inherent in each competent cell line, described below, will help you make the right choice so you can achieve your cloning goals. |
Choose Competent Cells for Your Application
Considerations when choosing competent cells for your application?
Transformation efficiency
The transformation efficiency you need will be largely determined by your application. To meet different cloning requirements, competent cells are available in a wide range of transformation efficiencies - from >1 × 106 for routine cloning to >3 × 1010 cfu/μg pUC DNA for library construction or working with limited amounts of DNA.
Figure 1. A range of transformation efficiencies are available to meet your application needs. One Shot® cells are provided at >5 x 109, 1 x 109, or >1 x 108 cfu/µg.
Determining transformation efficiency
Usually the transformation efficiency of competent cells is measured by transforming them with subsaturating amounts of supercoiled pUC DNA (~10–500 pg). The results are expressed in number of transformants (or colony forming units) per microgram of plasmid DNA (cfu/μg).
Transformation Efficiency Calculations
Transformation Efficiency Calculations
| # of colonies | x | 106 pg | x | 300 µl total transformation volume | = | # of transformants |
| 100 pg transformed DNA | µg | ? µl plated | µg plasmid DNA |
Troubleshooting your transformations
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 volume | Add 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 ligation | For 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 resistance | Store 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 cells | Thaw competent cells on ice, and use cells immediately upon thawing. Do not vortex. |
| Improper heat-shock procedure for chemically competent cells | For 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.
|
| Improper electroporation | Use devices that apply 16 kV/cm and the appropriate conditions for each electrocompetent strain |
| Slow or no growth of cells | If 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. |
