[Music] Thank you. The genomic DNA extraction kit provides a quick, simple, and less toxic method for isolating DNA from different types of biological samples. Extracted DNA is free of contaminants and is suitable for downstream applications like PCR, restriction enzyme digestion, cloning, and sequencing. DNA extraction kits employ silica-based technology, which allows the negatively charged DNA to bind to the silica membrane embedded in the spin column in the presence of high chaotropic salts. The objectives of this activity are to learn DNA extraction using a DNA extraction kit and to describe the principle behind the solid-phase extraction method. The materials include bacterial culture in broth media, 1.5 ml microcentrifuge tubes or Eppendorf tubes, microcentrifuge tube racks, refrigerated centrifuge, micropipettes, and pipette tips, ice bucket, lysozyme, RNase A, proteinase K, 95 to 100% ethanol, and DNA extraction kit. gDNA extraction using a commercial kit involves seven steps: 1. Cell collection, 2. Cell resuspension, 3. Cell lysis, 4. Preparation of spin column.

five binding of DNA to column 6. Washing of DNA bound to the column and seven illusion of extracted DNA for Step 1. Collect 1.5 ml of bacterial broad culture in microcentrifuge tubes and centrifuge at 12,000 G for two minutes. After centrifugation, discard the supernatant for Step 2. If working with gram-negative bacteria, resuspend the cell pellet in 180 microlit lysis solution. Step 2. For gram-positive bacteria, resuspend the cell pellet in 200 microlit lysozyme solution and incubate at 37 degrees Celsius for 30 minutes. Lysozyme will enhance the lysis of the bacterial cell wall by hydrolyzing the thick peptidoglycan layer in gram-positive bacteria. For Step 3, add 20 microlit proteinase K to the cell suspension. Vortex or pipette to mix. Incubate at 55 degrees Celsius for 30 minutes. This will degrade any contaminating proteins from the extract.

It will also deactivate RNases and DNases that would degrade the DNA. For step 4, add 500 microlit of the column preparation solution to each binding column. Centrifuge at 12000 G for one minute. Discard the flow-through. Step 5: add 200 microlit of ethanol to the lyse cells. Vortex or mix by inversion. DNA is insoluble in alcohol and thus will allow the DNA to precipitate. Transfer the ethanol mixture to the binding column. Centrifuge at 6500 G for one minute. For step 6, transfer the column into a new collection tube. Add 500 microlit of wash solution to the column. Centrifuge at 6500 G for one minute. Transfer the column to a new collection tube and add 500 microlit of wash solution concentrate to the column. Centrifuge at 12000 G for three minutes to dry the column. For the final step, transfer the column to a new collection tube. Add 200 microlit of elution solution. Centrifuge at 6500 G for one minute. This

will release the DNA bound in the silica column in the collection tube. Store gDNA in -20 or -80 degrees Celsius. This result shows gDNA extracted from three bacterial species using a DNA extraction kit. Lane 3 and 4 is from Bacillus cereus, and Lane 5 and 6 is from Micrococcus luteus. There is no visible gDNA degradation and contamination, which suggests high-quality DNA. [Music] Thank you.

Activity 3. Genomic DNA Extraction using DNA Extraction Kit (Solid-Phase Extraction Method)

Activity 3. Genomic DNA Extraction using DNA Extraction Kit (Solid-Phase Extraction Method)

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