Contents
Preparation of CaCl2 competent cells
- Dilute 500μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells.
- Incubate at 37⁰C, shaking at 225rpm for 105 minutes.
- Spin down for 2 minutes at 7000G at 4⁰C.
- Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice.
- Repeat centrifugation for 2 minutes at 7000G at 4⁰C.
- Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice.
- CaCl2 competent cells can be kept on ice in the fridge overnight.
Transformation of CaCl2 competent cells
- Add 1μl of plasmid DNA to 100μl of competent cells.
- Heat shock at 42oC for 45 seconds.
- Add 200μl of L-broth of the sample.
- Keep on ice for 2 minutes.
- Incubate the cells at 37oC. The time varies depending on which antibiotic resistance the plasmid holds.
Figure 1: Incubation time for transformed cells containing different antibiotic resistances
Restriction digests
A 20μl reaction typically contained:
- 2μl buffer
- 4μl Miniprep (or 8μl G-Block) dependant on concentration of Miniprep
- Make up to 20μl with ddH20
- Vortex briefly.
- Incubate at 37oC for at least 60 minutes.
- Heat inactivate restriction digests where appropriate.
Materials and Methods
Condition set up
Sample preparation
- 1
- 2
- 3
Table 1: Optical density analysis of S. thermophilus growth
Results and Discussion
Outlook
References
- ↑ Kiliç, A. O., Pavlova, S. I., Ma, W. G. & Tao, L. 1996. Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt. Appl Environ Microbiol, 62, 2111-6.
