Competent Cell Protocols
Introduction
Transformation is a process by which some bacteria take up foreign genetic material (naked DNA) from the environment. Once it enters the cytoplasm, the genetic material may be degraded by nucleases if it is different from the bacterial DNA. If the exogenous genetic material is similar to bacterial DNA, it may be integrated into the chromosome. Sometimes the exogenous genetic material may co-exist as a plasmid with chromosomal DNA.
Not all bacteria are capable of taking up exogenous DNA from their environment. For the bacterium to uptake DNA, it has to be competent. Bacteria can be naturally competent or made competent by artificial methods. The factors that regulate natural competence vary between various genera. The practical approach to acquire competent cells is to make the bacterial cells artificially competent using chemicals or electrical pulses.
- Chemical induction of competence involves the following steps:
- chilling the cells in the presence of calcium phosphate (Catalog No. CAPHOS) to make them permeable
- incubation with DNA
- heat shock treatment at 42 °C for 60-120 seconds that causes the DNA to enter the cells
Note: To endure the heat shock treatment, it is important the cells used are in the log phase of growth.
- Alternatively, the bacterial cells are made permeable by subjecting them to electrical pulses, a process known as electroporation.
The phenomenon of transformation has been widely used in molecular biology. Bacteria may be used as host cells to make copies of the DNA, cloning, to express large amounts of proteins, generation of cDNA libraries and in DNA linkage studies as they are easily grown in large numbers.
Competent cells available in our catalog
We offer a range of Escherichia coli bacterial cells made competent with the highest efficiencies by optimized procedure specific to each strain. Choose from 24 new competent cells for a wide variety of applications, including protein expression, routine or difficult cloning, and library generation. Many trial sizes available!
Protocols
Things to do before starting transformation:
- Prepare LB agar plates and allow to gel. If pre-poured plates are being used, ensure the plates are warmed to 37 °C.
- Depending on the antibiotic marker present in the plasmid DNA, incorporate appropriate antibiotic in the LB agar.
- If blue/white screening for recombinants is required, incorporate 1mM IPTG, 300µg/ml S-Gal or 40µg/ml X-Gal and 500µg/ml ferric ammonium citrate in the LB agar.
- If electrocompetent cells are being used, place the electroporation chamber on ice.
- Heat the water bath to 37 °C.
- Warm the sterile SOC medium to room temperature (or 20-25 °C in water bath).
Protocol for Transformation Using Chemically Competent Cells
Materials required but not provided:
Standard Transformation Protocol:
- Transfer the required number of tubes from -70 °C freezer to wet ice. Include an extra tube for control DNA, if desired.
- Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension.
- Add 1-50ng of purified plasmid DNA directly to cells in rest of the tubes. Mix by gentle tapping and place on ice.
Note: For control: Add 1µL (10ng) pUC19 control DNA to one tube. Mix by gentle tapping and place on ice. - Incubate the cells on ice for 30 minutes.
- Transfer the cells to 37 °C water bath for exactly 45 seconds.
- Transfer the cells to ice for 2 minutes.
- Add SOC medium to each tube. Transfer the cells to sterile polypropylene tubes and loosen the caps to facilitate aeration of the cultures.
- Incubates the cells on shaker incubator (225-250 rpm) at 37 °C for 1 hour.
- Pipette 10-100µL of each transformed cell suspension onto LB agar plates with selection antibiotic and spread it using sterile spreader.
- Incubate plates at 37 °C overnight.
- Select colony (colonies) and culture as needed.
- Isolate the plasmid DNA from each culture.
- Digest the plasmid DNA using restriction enzymes; separate by gel electrophoresis.
- Culture the preferred clones.
Protocol for transformation using Electrocompetent cells
Materials required but not provided:
Standard Transformation Protocol:
- Transfer the required number of tubes from -70 °C freezer to wet ice. Include an extra tube for control DNA, if desired.
- Allow the cells to thaw for 5 minutes. Gently tap the tubes multiple times to obtain uniform suspension.
- Transfer SOC medium to culture tubes, one for each transformation reaction, and leave at room temperature.
Note: The volume of SOC medium depends on the volume of cells that will be added in the next step. The final volume with the competent cells and SOC medium should be 1000µL. - Place 1mm standard cuvettes and sterile microcentrifuge tubes on ice, one for each transformation reaction.
- Transfer the competent cells to chilled microcentrifuge tubes. Use 40µL of cells from 80µL package and 50µL of cells from 100µL package.
- Prepare 5-fold dilution of DNA or ligation mix in TE buffer. Add to microfuge tubes.
Note: For control: Add 1µL of 5-fold dilution of pUC19 control DNA to one tube. - Pipette the DNA and cells mixture into chilled 1mm cuvette.
- Set electroporator to a field strength of 25kV/cm for 6ms and treat the cells.
- Remove cells from cuvettes and add to tubes containing SOC medium.
- Incubate the cells on shaker incubator (225-250rpm) at 37 °C for 1 hour.
- Pipette 10-100µL of each transformed cell suspension onto LB agar plates with selection antibiotic and spread it using sterile spreader.
- Incubate plates at 37 °C overnight.
- Select a colony and culture as needed.
- Isolate the plasmid DNA from each culture.
- Digest the plasmid DNA using restriction enzymes and separate by gel electrophoresis.
- Culture the preferred clones.
Important Tips for Optimization of Transformation Process:
- Confirm the cells are still frozen and dry ice is still present upon receipt.
- For maximum transformation efficiency, use high quality DNA sample free of phenol, ethanol, proteins, salts or detergents. When using electrocompetent cells, high salt content in DNA will result in arcing at high voltage, which may damage the sample and the equipment.
- DNA in ligation reactions containing high quality reagents may be used for transformation. The DNA ligase need not be inactivated.
- Keep the competent cells on ice at all times; prevent accidental warming of the cells.
- Tap the tubes gently to obtain uniform suspension of cells. Do not mix by using vortex or pipette.
- Warm LB agar plates to 37 °C for optimal colony growth.
- Electroporator settings for transformation using electrocompetent cells:
- BTX Model ECM 630: HV mode, 2.5kV, 25µF, 100Ω, 1mm cuvette
- BioRad Gene Pulser: 2.5kV, 25µF, 100Ω, 1mm cuvette
References
To continue reading please sign in or create an account.
Don't Have An Account?