In this lab, I am going to use antibiotic-resistance plasmids to transform Escherichia coli.
For this lab you will need the following:
Sensitive E. coli (-ampR)
Water bath to heat shock cells
A freezer to incubate cells
Step 1: Wash hands and sanitize lab setting. This will prevent anything reacting with a substance that could have been present when it shouldn’t have been. Step 2: Ampicillin sensitive E. coli cells in log phase of growth are transferred to cold CaCl2 solution. Step 3: ampR plasmids are added to experimental cells only. Step 4: Cells are heat-shocked at 42oC. Some of the competent cells take up the ampR plasmid and are transformed. Step 5: The treated cells are spread on an agar plate containing ampicillin. Ampicillin kills the cells that lack the ampR gene. Step 6: The cells are incubated for 24 hours.
Step 7: Only colonies of E. coli that have been transformed by the ampR gene will grow.
All E. coli cells transferred. There were colonies on the perti dishes. No lawns were present.
In this experiment, we learned about genetic transformation. This is when a host organism takes in foreign DNA and expresses the foreign gene. We used E. coli in this lab because it grows very rapidly. We used plasmids to enter the foreign DNA into the cells. We then made our cells competent by a process that uses calcium chloride and heat shock. We learned that there is no ampicillin in the LB agar because E. coli, which is sensitive to the ampicillin, covered the plate with a lawn of cells. The reason we used LB (Luria Broth) is because it is food, and it will help the bacteria grow. We added ampR plasmids to the experimental cells and heat-shocked them. When the cells were spread on the LB agar containing ampicillin and incubated for 24...
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