Cloning & Expression
Calculating Transformation Efficiency
Transformation efficiency is the number of transformed cells (transformants) generated by 1 µg of supercoiled plasmid DNA in a transformation reaction. A known quantity of pUC19 DNA is typically used as the control.
Transformation efficiency (transformants/µg) is calculated as follows:
# colonies on plate/ng of DNA plated X 1000 ng/µg
How much DNA is plated? 0.1 ng of control DNA (1 µL of 0.1 ng/µl, freshly diluted) is added to 100 µL of competent cells. 900 µL of SOC medium is added prior to expression. 10 µL (equivalent to 0.001 ng DNA) is then diluted in 990 µL SOC and 100 µL is plated (equivalent to 0.0001 ng DNA).
If 100 colonies are counted on the plate, the transformation efficiency is:
100 transformants / 0.0001 ng X 1000 ng/µg = 1 x 109 transformants/µg.
Factors That Affect Transformation Efficiency
- 1. DNA
- Actual DNA Concentration
The amount of pUC19 DNA in the control tube included with Sigma-Aldrich competent cells is carefully quantitated. Other vendors may have different amounts of plasmid DNA in their preparations. Therefore, when comparing competent cells, it is advisable to use the same DNA for all transformations.
Forms of DNA
Linear and single-stranded DNA transforms <1% as efficiently as supercoiled DNA.
Purity of DNA
- For electroporation, never use more than 1 µl plasmid DNA per transformation. The salts contributed by the preparation can cause low transformation efficiencies.
- Column-purified DNA is generally free of contaminants that would interfere with chemical transformation.
- Contaminants in miniprep DNA can interfere with transformation. Limit the amount of miniprep DNA in a transformation as much as possible and never use more than 5 µl per 50-µl reaction.
- Ligation mixtures inhibit transformation. Ligase strongly inhibits electroporation, but this effect can be limited by heat inactivating the ligase in the ligation mixture (65°C for 5 min) prior to adding DNA to the cells.
Amount of DNA
The transformation reaction is generally saturated at >10 ng DNA. We use 0.1 ng pUC19 in the control reaction.
- 2. Heat Shock
- The heat shock step can affect transformation efficiency. Optimal efficiencies are obtained with a thin-wall tube such as a PCR tube or another thin-walled tube (42°C for 45 sec). With thick-walled tubes, such as a microcentrifuge tube, the optimal parameters are slightly different (37°C for 60 sec). When in doubt, it is better to use the 60 second shock at 37°C.
- 3. Length of Time After Transformation
- The effect of the expression time depends on the plasmid and the strain. With pUC19 and GC5, the efficiency is down ten-fold if the cells are plated without any expression time at all, down seven-fold if plated after fifteen minutes, and down three-fold if plated after thirty minutes. Longer expression times give higher numbers of colonies, but since they are siblings, the number of transformants is not higher.
- 4. SOC Medium
- SOC Medium is highly recommended as the expression medium. SOC Medium gives two-fold better results than LB Medium for chemically competent cells.
- 5. Selective Plates
- Some batches of commercially prepared agar plates are better than others. Plating large numbers of cells on selective plates or for extended periods of incubation may allow growth of satellite colonies. The colonies can be identified by streaking them on the selective agar plates. If they are true transformants, they will grow. If they are satellites, they will not grow.
- 6. Freeze/Thawing of Cells
- Cells that are refrozen will lose activity, typically at least two-fold.
Back to top
Back to Cloning and Expression