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, as it allows the introduction of foreign DNA into bacterial cells.Experimental Principle
Competent cells are those that are in a state where they can efficiently take up exogenous DNA. In this procedure, *E. coli* cells were suspended in a cold CaCl₂ hypotonic solution at 0°C, causing the cells to swell and become more permeable. The DNA forms a calcium phosphate complex with the CaCl₂, which then adheres to the cell membrane. A short heat shock at 42°C is applied to facilitate the uptake of the DNA by the cells, allowing them to become competent for transformation.Instruments, Materials and Reagents
The following instruments and materials were used: 1. Ultra-clean laminar flow hood 2. Low-temperature centrifuge 3. Incubator shaker (set at 37°C, 230 rpm) 4. Autoclave 5. Water bath set at 37°C 6. Petri dishes **Materials and Reagents:** 1. *E. coli* strain DH5α 2. 0.1 M CaCl₂ solution (sterilized by filtration or autoclaving) 3. LB broth: 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 the liquid LB medium as above, add 16–18 g agar before autoclaving.Experimental Procedure
1. Inoculate a single colony of *E. coli* DH5α into 20 mL LB broth and incubate overnight at 37°C with shaking. 2. Using a 1% inoculation ratio, transfer 20 mL of fresh LB broth and incubate at 37°C with shaking at 230 rpm for 3 hours. 3. Take 1 mL of the culture, centrifuge at 4,000 rpm for 3 minutes at 4°C, 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 at 4°C, and remove the supernatant. 5. Add 100 µL of ice-cold 0.1 M CaCl₂, resuspend the pellet, and centrifuge at 4,000 rpm for 4 minutes at 4°C. Discard the supernatant. 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 experiments. By carefully following these steps, researchers can effectively produce competent bacterial cells, which serve as an important tool in genetic engineering and molecular biology.Brazilian Human Hair Wig,Wigs For Black Women,Curly 100% Unprocessed Virgin Brazilian Deep Wave
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