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Preparation
Some basic steps can be taken to expedite the overall TOPO cloning process and help ensure its success. First, tubes of viable cells should be placed on ice to maintain their acceptability. Next, the super optimal culture (SOC) medium, bacterial growth enriched with nutrients, should be brought to room temperature. The selective plates should then be pre-warmed to 37 degrees Celsius so they are fully ready when it comes time to use them.
Combining Ingredients
To begin the cloning process, a combination of 2 microliters of polymerase chain reaction (PCR) product, 0.5 microliters of salt solution, and 0.5 microliters of cloning vector should be combined in a 0.5-milliliter centrifuge tube. Once combined, these ingredients will need to be set at room temperature and allowed to incubate for five to ten minutes. Next, the combined ingredients will be added to "Top-10 chemically competent cells" in a 50-microliter tube and placed on ice for an additional ten minutes. After this, the cells must be heat shocked at 42 degree Celsius exactly and placed on ice for an additional minute before 180 microliters of the SOC medium is added. This mixture should then be shaken in a horizontal direction at 200 rpm for an additional hour at 37 degrees Celsius.
Final Steps
A mixture of 50 microliters and 100 microliters of each reaction should be plated out "onto LB + AMP + X-Gal plates and incubate without shaking overnight at 37 C." The next day the three white colonies from each reaction should be picked out and placed in 4 ml of LB + AMP and left to incubate overnight at a temperature of 37 degrees Celsius while being shaken at 200 rpm. The final step is the mini-preparation of the plasmids and waiting to see whether the process was successful.