Purpose
This experiment aimed to successfully prepare competent *E. coli* cells, which are essential for genetic transformation experiments. Understanding and mastering this technique is crucial for molecular biology research, especially when introducing foreign DNA into bacterial cells.Experimental Principle
Competent cells are bacteria that are in a physiological state that allows them to take up exogenous DNA. In this experiment, the cells were treated with calcium chloride (CaCl₂) under hypotonic conditions at 0°C, causing the cell membranes to swell and become more permeable. The DNA in the transformation mixture forms a complex with calcium ions, creating a stable structure that can adhere to the cell surface. A brief heat shock at 42°C is then applied to facilitate the uptake of the DNA by the cells, enabling successful transformation.Materials, Instruments, and Reagents
- **Instruments**: 1. Ultra-clean laminar flow hood 2. Low-temperature centrifuge 3. Incubator shaker (37°C, 230 rpm) 4. Autoclave 5. Constant temperature water bath 6. Petri dishes - **Materials and Reagents**: 1. *E. coli* strain DH5α 2. 0.1 M CaCl₂ solution (sterilized on ice) 3. LB liquid medium: Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water. Adjust pH to 7.0 using 5 M NaOH, then bring to 1 L with deionized water. Sterilize by autoclaving at 121°C for 20 minutes. 4. LB agar: Prepare as above, but add 16–18 g agar before sterilization.Experimental Procedure
1. Inoculate a single colony of *E. coli* DH5α into 20 mL of LB medium and incubate overnight at 37°C with shaking. 2. Inoculate 20 mL of fresh LB medium with 1% of the overnight culture and incubate at 37°C with shaking at 230 rpm for 3 hours. 3. Take 1 mL of the culture, centrifuge at 4°C, 4,000 rpm for 3 minutes, and discard the supernatant. 4. Resuspend the pellet in 500 µL of ice-cold 0.1 M CaCl₂, centrifuge again at 4,000 rpm for 4 minutes, and remove the supernatant. 5. Add 100 µL of ice-cold 0.1 M CaCl₂, resuspend the pellet, and centrifuge for another 4 minutes at 4°C. 6. Finally, resuspend the pellet in 50 µL of ice-cold 0.1 M CaCl₂ and incubate on ice for 3–24 hours. These are now competent *E. coli* cells ready for transformation. This detailed procedure ensures that the cells are in the optimal condition for efficient DNA uptake, making it a fundamental step in many genetic engineering workflows.Deep Wave Wig Transparent 13x4 13x6 Hd,Deep Wave Wig Hd Lace,Deep Wave Frontal Wig Human Hair
Xu Chang Zhuo YunQing crafts Co., LTD , https://www.wowqueenhair.com