Competent Cell Transformation Efficiency Optimization
by Jennifer Hornstein - 04/11/12
Worried about seeing lower efficiency from your competent cell transformation than you expected? There are many reasons that efficiency can be less than optimal. Below we will discuss the basics to help you learn more about what to avoid to see maximum efficiency.
First, let's start with what transformation efficiency is and how to calculate it. Then we will explore what factors affect transformation efficiency.
What is transformation efficiency?
The competence of a microorganism is dependent on its ability to uptake recombinant DNA and survive the introduction of foreign DNA into the cell. Different organisms vary in these capacities, however the basic principles of introduction are the same. Modifications to the cell membrane/wall of microorganisms must occur often, using either chemical modification or electric shock.
How to calculate transformation efficiency
After the above damage, cells are recovered and calculated for their “uptake” efficiency—measured as colony forming units per microgram of DNA (cfu/μg).
To maximize efficiency from the onset or to troubleshoot low efficiency results, use the below competent cell transformation troubleshooting tips and suggestions.
Factors that affect transformation efficiency:
1. 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).
2. 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.
3. 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.
4. 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
5. Improper handling of competent cells - Thaw competent cells on ice, and use cells immediately upon thawing. Do not vortex.
6. 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. If there is a change in the tubes or volume of cells, the heat shock conditions must be optimized.
7. Improper electroporation
Use devices that apply 16 kV/cm and the appropriate conditions for each electrocompetent strain
8. 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.
9. 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.
- A researchers guide to cloning DNA
- Cloning and transformation
- Transformation cloning efficiency
- Chemically competent cells
- Electrocompetent cells
For further assistance, contact Life Technologies technical support to continue troubleshooting your competent cell transformation.