My internship in laboratory at 2025.11.7
Description
Morning: Learned Streak Plate Technique Transferred a microbial colony from one culture dish to another to dilute the population and obtain isolated pure colonies for DNA sequencing. Procedure: 1. Sterilized tools. 2. Inside a UV cabinet, used a sterilized inoculation loop to scrape a small amount of the microbial colony. Streaked tight zigzag lines in the first quadrant (approximately 1/4 of the new, sterile culture dish). 3. Sterilized the inoculation loop by flaming it in an alcohol burner until the wire tip glowed red and the rest passed through the flame. Allowed it to cool until it could briefly touch the edge of the medium without melting it to prevent killing microorganisms. 4. Dragged the loop through the first quadrant streaks and streaked several new lines in the adjacent second quadrant without touching the first area. 5. Re-sterilized and cooled the loop again, then streaked the third quadrant. 6. Repeated sterilization and cooling, then streaked the fourth quadrant (where isolated single colonies are most likely to appear). 7. Inverted the culture dish to prevent condensation from disrupting the colonies. 8. Placed the dish in an incubator and cleaned up the work area. Note: The culture medium contained nitrogen sources, carbon sources, and sodium chloride (essential environment for microbial growth). --- Afternoon: Observed Bacterial Genomic DNA Extraction Experiment specific procedures are described below
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Procedures of Bacterial Genomic DNA Extraction Experiment 1. Transferred bacterial suspension to a 1.5 mL centrifuge tube. Centrifuged at 10,000 rpm for 1 minute and carefully removed the supernatant. 2. Added Buffer GA and vortexed until the pellet was fully suspended. 3. Added Proteinase K solution and mixed thoroughly (digests proteins, releasing and protecting DNA). 4. Added Buffer GB, vortexed for 15 seconds, and incubated at 70°C for 10 minutes (separates proteins from DNA, achieving complete cell lysis). 5. Added absolute ethanol and vortexed for 15 seconds (creates a high-salt/alcohol environment essential for DNA binding to the silica membrane). 6. Placed the mini adsorption column CB3 into a collection tube. Transferred the resulting mixture into the column and centrifuged for 30 seconds (liquid passes through the silica membrane; DNA binds specifically to the membrane under high-salt conditions, while proteins, cellular debris, and other impurities pass through as waste). Note: DNA carries a negative charge. The high-salt environment neutralizes these charges, reducing electrostatic repulsion between DNA molecules. Cations in the high-salt buffer "shield" the negative charges on both the silica membrane and the DNA, allowing adsorption via hydrogen bonding and van der Waals forces. 7. Added Buffer GD to the adsorption column, centrifuged for 30 seconds, discarded the flow-through, and placed the column back into the collection tube (further washes residual protein impurities from the membrane). 8. Added Wash Buffer PW (contains ethanol and buffer salts) to the adsorption column, centrifuged for 30 seconds, discarded the flow-through, and returned the column to the collection tube (thoroughly removes residual salts; pure DNA remains bound to the silica membrane). 9. Repeated the wash step. 10. Performed a high-speed spin to remove residual liquid. Air-dried at room temperature for 2-5 minutes to evaporate trace ethanol completely (ethanol can interfere with subsequent enzymatic reactions). 11. Transferred the adsorption column to a clean collection tube. Added Elution Buffer TE dropwise directly onto the center of the membrane without touching it. Let stand at room temperature for 2-5 minutes to allow the buffer to fully hydrate the membrane and dissolve the DNA. Centrifuged for 2 minutes; the liquid collected at the bottom of the tube is the purified genomic DNA. The standard protocol should include verification of purity and concentration, but this was not performed as the lab lacked the capability, so the process concluded here. Concentration verification method: Agarose gel electrophoresis. Purity verification method: Ultraviolet spectrophotometry.