DNA is inserted into bacterial host cells through a process known as transformation. The easiest type of transformation is heat shock. This process involves briefly exposing the bacteria to warm temperature. This stresses the cells and allows small molecules, such as DNA, to enter. The bacterial cells then contain the DNA and copy it, along with their own DNA, when they divide. They will also make the protein coded for by the DNA. This method can be used to make proteins for research, as well as for clinical purposes to treat disease.
Place approximately 50 microliters of competent cells in a microcentrifuge tube. Competent cells are bacterial cells that easily take in DNA through transformation. Make sure the tube is on ice. Leave the tube on ice for ten minutes to allow enough time for the cells to thaw. Make sure the cells are completely thawed before proceeding.
Add 50 nanograms of the DNA you want to insert.
Transfer the tube containing the competent cells and the DNA into a water bath set at 42 degrees Celsius (107.6 degrees Fahrenheit). Hold the tube in the water bath for exactly 45 seconds. Immediately place the tube back on ice and hold it there for two minutes. The short heating time will stress the cells and cause them to take in the DNA. Placing the cells back on ice immediately will allow them to recover.
Add one milliliter of bacterial growth medium to the tube after the cells have recovered. Make sure that the cells are well mixed in the medium by inverting the tube or by gently pipetting up and down.
Place the cells in an incubator set at 37 degrees Celsius (98.6 degrees Fahrenheit) for 30 to 60 minutes. Make sure the tube is gently agitated during this entire time to prevent the cells from settling to the bottom of the tube.
Dip a cell spreader in alcohol and run through an open flame from a Bunsen burner to sterilize the spreader and minimize the risk of contamination from other sources of bacteria.
Take 100 microliters of the cell suspension containing bacterial growth medium and pipette onto an agar plate. Spread the solution evenly over the plate with the cell spreader. If the DNA inserted into the cells was in a plasmid that contained a gene for antibiotic resistance, such as ampicillin resistance, be sure that the agar plates contain that antibiotic. This will kill the cells that did not take in the resistant DNA.
Place the agar plates in a 37 degrees Celsius (98.6 degrees Fahrenheit) incubator overnight. The next morning, the plates will have bacterial cells containing the inserted DNA growing on them. This bacteria can then be transferred from the plate into a larger volume of bacterial growth medium and incubated again overnight if more of the bacteria are needed.
- Make sure all the tubes are chilled on ice prior to beginning the procedure.
- If the DNA is in plasmid form, it is best if the plasmid contains a gene for antibiotic resistance. This will allow you to selectively grow only the cells that took in the plasmid when grown on plates containing the antibiotic.
- Competent cells are bacterial cells. They are a biohazard and care should be taken when handling and disposing of the cells or any materials that come in contact with the cells.
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