Before you begin your work you should put on personal protective equipment as deemed appropriate by your school. These may include
- Nitrile gloves
- Eye protection and
- A lab coat.
1. Begin by labeling 1 small eppendorf tube “Cells”
2. Pipet 200 ul of CaCl2 into the eppendorf. This can be done by measuring 100 ul of solution twice. Place the tube on ice.
3. Use a sterile wooden dowel to scrape up one entire patch of cells, NOT including the agar that they’re growing on! Swirl the cells into the tube of cold CaCl2. NOTE: A small bit of agar can get transferred without consequence to your experiment, but remember you’re trying to move the cells to the CaCl2, not the media they’re growing on.
4. If you have a vortex, you can resuspend the cells by vortexing very briefly. If no vortex is available, gently flick and invert the eppendorf tube, then return it to the ice. Your teacher will explain how this step helps to make the bacterial membrane porous and how the cells are fragile at this point.
5. Label 2 small eppendorf tubes either “No DNA” or “+DNA”
6. Pipet 5 ul of DNA, called pBNAN, into the eppenforf tube labelled “+DNA.”
7. Place both the “No DNA” and the “+DNA” tube on ice
8. Find the “Cells” tube in your ice bucket and flick the tube with the competent cells to mix them.
9. Pipet 100 ul of the bacteria into the tube labeled “+DNA” and the remaining volume of cells into the tube labeled “No DNA.” NOTE: There may not be precisely 100 ul to add to the second tube and that’s OK.
10. Flick to mix the DNA and the cells in these tubes and return them to the ice for 2 minutes. You can use a timer or the clock to keep track of this incubation time.
11. While your DNA and cells are incubating, you can label the bottoms (not the tops) of the 2 petri dishes you’ll need. The label should indicate the name of the E.coli strain you’ve used (NB370) and the DNA you’ve transformed them with (“No DNA” or “pBNAN”). You should also put on your name or initials and today’s date.
12. Heat shock all of your DNA/cell samples by placing the tubes at 42° for 90 seconds exactly (use a timer or the clock). Your teacher will explain how this step helps drive the DNA into the cells and closes the porous bacterial membranes of the bacteria.
13. At the end of the 90 seconds, move the tubes to a rack at room temperature.
Pipet 500 ul of room temperature LB into each of the tubes (“No DNA” “+pBNAN”). Close the caps, and invert the tubes to mix the contents.
14. Using a sterilized spreader, spread 100 ul of the transformation mixes onto the surface of LB+ampicillin agar petri dishes.
15. If desired the remaining volumes of transformation mixes can be plated on LB plates to show the effect of antibiotic selection on the outcome. What would you expect from this experiment if you did it?
16. Incubate the petri dishes with the agar side up at 37° overnight, not more than 24 hours– or at room temperature at least 2 days.