In this exercise, you will learn how E. coli cells can be transformed by foreign plasmid DNA. The exercise uses frozen E. coli cells that have been prepared in a certain way to become transformation competent. The theory is that the preparation of the cells reduce the integrity of the cell membrane, and allows plasmid DNA to diffuse into the cell
Learning Objectives
At the end of the activity, you will be able to:
Abilities
Understand the theory behind bacterial transformations
Perform a bacterial transformation using E.coli cells
Skills
Accurate pipetting
Performing a bacterial transformation
Spotting and streaking cells on agar plates
Determining proper controls and antibiotic selections.
Instructions
For this experiment we will be doing two transformations using the ligation product we produced in Exercise 9. We will be doing another transformation with a plasmid that we know has been successfully been transformed in the past from our samples in the freezer (your positive control).
1. Retrieve the appropriate number of tubes containing competent E. coli cells from the -80°C freezer. Place the tubes in the ice bucket and thaw for 10-15 min.
2. Turn on your flame before opening the tubes and ensure the following steps are done in a sterile environment (See exercise 1).
3. On ice, mix 50µL of competent cells and 2µl of the plasmid DNA. The concentration of plasmid DNA should be in the 20-200 ng/µL range, - mix with pipette
4. Incubate on ice for 20 minutes
5. Heat shock at 42Ëš for 45 seconds. Return to ice.
6. Add 1ml of LB media. Under a flame with sterile technique
7. Move tubes to 37Ëš - rolling or shaking it best, but just sitting in a rack is OK. Incubate for 1 hr.
8. Spin down cells at in a microfuge at top speed for 10-30 seconds.
Turn on your flame for the next steps
9. Remove all but ~50µl of the media using a pipette.
10. Resuspend the cells by pipetting up and down, and plate on LB + selection. (Selection is the antibiotic resistance of your plasmid). To plate the cell suspension, just take it up with a pipette and pipette it into the middle of the plate.
11. Retrieve a vial of beads and add approximately 6-10 beads to your plate. Make sure all the beads have passed through the sample in the middle of the plate by tilting the plate.
12. Now shake your plate on the lab bench to ensure that the cell suspension is spread over the plate
13. Put the plates upside down (agar side up) in the 37°C incubator.
14. Incubate for 14-18 hours until distinct colonies are clearly visible. Do not overgrow. Selection may be lost over time as the antibiotic starts to break down.
Note-- if it is your first time conducting a bacterial transformation, do the experiment with a positive and a negative control. The positive and negative controls. The positive control experiment is a transformation with stock DNA plasmid. Your negative control experiment is a mock transformation with water
