Chemically competent cells are a suitable solution for conventional cloning and subcloning experiments. Treatment with calcium chloride promotes the adhesion of plasmid DNA to the competent cell membrane. Heat shock the sensory cells through a water bath to open the cell membrane pores, allowing plasmids to enter.
Operation method: (The following operations are carried out according to the standards of sterile conditions)
1. Take the competent cells and place them in an ice bath. If necessary, transfer the freshly melted cell suspension into sterile pre cooled centrifuge tubes and place them in an ice bath. The recommended dosage for converting competent cells at once is 50-100 μ l, which can be divided and used according to the actual situation. It should be noted that the volume of DNA used should not exceed one tenth of the volume of the competent cell suspension.
The following experiment takes 100 μ l of competent cells as an example.
2. Add the target DNA (50 μ l of competent cells can be saturated with 1ng of supercoiled plasmid DNA) to the suspension of competent cells, gently rotate the centrifuge tube to mix the contents, and let it stand in an ice bath for 30 minutes.
3. Place the centrifuge tube in a 42 ℃ water bath for 90 seconds, then quickly transfer the tube to an ice bath to cool the cells for 2-3 minutes without shaking the centrifuge tube.
4. Add 500 μ l of sterile SOC or LB medium (without antibiotics) to each centrifuge tube, mix well, and shake at 37 ℃ for 45 minutes (150 revolutions per minute) to induce expression of resistance marker genes on the plasmid and revive the bacterial cells.
5. Mix the contents of the centrifuge tube evenly, take 100 μ l of transformed competent cells and add them to SOB or LB solid agar medium containing the corresponding antibiotics. Gently spread the cells evenly with a sterile curved glass rod. Place the plate at room temperature until the liquid is absorbed, invert the plate, and incubate at 37 ℃ for 12-16 hours.
Note: The amount of coating can be adjusted according to specific experiments. If the total amount of transformed DNA is large, a smaller amount of transformed product can be coated onto a plate; On the contrary, if the total amount of transformed DNA is small, 200-300 μ l of transformed product can be coated onto a plate. If there are fewer expected clones, a portion of the culture medium can be removed by centrifugation (4000rpm, 2 minutes), and the bacterial cells can be suspended and coated on a plate. (The remaining bacterial solution can be stored at 4 ℃. If the number of transformed colonies on the next day is too low, the remaining bacterial solution can be coated on a new culture plate.)
Notes:
When performing conversion operations, they should be carried out according to the requirements of the corresponding temperature and sterile conditions.
2. Sensory cells should be stored at -70 ℃ and should not be frozen or thawed multiple times or left for too long to avoid the transformation efficiency of competent cells.
To prevent unsuccessful conversion experiments, a portion of the connecting reaction solution can be retained for re conversion, reducing losses to a very low level.