Plasmid DNA Isolation Lab 7 PDF

Plasmid DNA Isolation Biochemical and Genetic Techniques Lab 7 Three stage  Plasmids Definition: Plasmid is a double-stranded, circular extrachromosomal DNA found in bacteria. Function: Used in recombinant DN...

Plasmid DNA Isolation Biochemical and Genetic Techniques Lab 7 Three stage  Plasmids Definition: Plasmid is a double-stranded, circular extrachromosomal DNA found in bacteria. Function: Used in recombinant DNA experiments. Applications: Used for cloning genes from other organisms. Enables the production of large quantities of foreign DNA. Transfer: Plasmids can be transferred between bacteria. Transfer can occur within the same species or between different species. Size: Plasmids range in size from 1 to 1000 kilo base pairs. Comparison with Chromosomal DNA: Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium. Bacterial chromosomal DNA can contain several million base pairs. Alkaline Lysis Method for Plasmid DNA Extraction: Cell Growth and Harvesting: Bacterial cell culture containing the plasmid is grown to sufficient density. Cells are pelleted through centrifugation to separate them from the growth medium. Re-suspension: The cell pellet is re-suspended in a solution (solution I) containing Tris, EDTA, glucose, and RNase. Divalent cations (Mg2+, Ca2+) are crucial for DNase activity and cell wall integrity. EDTA chelates divalent cations, preventing DNase damage and destabilizing the cell wall. Glucose maintains osmotic pressure to prevent cell bursting, and RNase degrades cellular RNA. Lysis: Lysis buffer (solution II) contains sodium hydroxide (NaOH) and the detergent SDS. SDS solubilizes the cell membrane, while NaOH breaks down the cell wall and denatures DNA. Denaturation converts double-stranded DNA (dsDNA), including plasmid and genomic DNA, into single-stranded DNA (ssDNA). Vigorous mixing should be avoided to prevent shearing of genomic DNA.  Neutralization: Addition of potassium acetate (solution III) decreases alkalinity. Hydrogen bonding between bases of ssDNA is re-established, allowing selective re- naturation of plasmid DNA. Vigorous mixing is avoided to prevent re-annealing of sheared genomic DNA. Separation and Cleaning: Precipitation of denatured cellular proteins, SDS, and single-stranded genomic DNA occurs. Centrifugation separates this white precipitate from the plasmid DNA solution. Cleaning and Concentration: Plasmid DNA, though separated from cell debris, remains in a solution with salt, EDTA, RNase, and residual cellular proteins. Cleaning and concentration methods, such as phenol/chloroform extraction followed by ethanol precipitation, are employed. Procedure: 1. Inoculation: 1. Inoculate 2 ml of rich medium (broth) with the appropriate antibiotic using a single colony of transformed bacteria. 2. Incubate the culture overnight at 37°C with vigorous shaking. 2. Centrifugation: 1. Pour 1.5 ml of the overnight culture into a microfuge tube. 2. Centrifuge at maximum speed for 30 seconds at 4°C to pellet the bacteria. 3. Medium Removal: 1. Aspirate the medium, leaving the bacterial pellet as dry as possible. 4. Resuspension: 1. Vigorously vortex the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I. 5. Alkaline Lysis: Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly and invert to mix well. Do not vortex. Store the tube in ice. 6.Neutralization: 1. Add 150 μl of ice-cold Alkaline lysis solution III. 2. Close the tube and disperse Alkaline lysis solution III by inverting the tube several times. 3. Store the tube in ice for 3-5 minutes. 7.Centrifugation: 1. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C. 2. Collect the supernatant into a fresh tube. 8.(Optional) Phenol-Chloroform Extraction: 1. Optionally, add an equal volume of phenol:chloroform. 2. Mix the organic and aqueous phases by vortexing and then centrifuge at maximum speed for 2 minutes at 4°C. 3. Transfer the aqueous upper layer to a fresh tube. 9.Nucleic Acid Precipitation: 1. Add 2 volumes of ethanol at room temperature to precipitate nucleic acids. 2. Vortex the solution and let it stand for 2 minutes at room temperature. 3. Centrifuge at maximum speed for 5 minutes at 4°C. 10.Supernatant Removal: 1. Aspirate the supernatant and invert the tube to drain any remaining fluid. 11.Ethanol Wash: 1. Add 1 ml of 70% ethanol to the pellet, invert the closed tube, and recover DNA by centrifugation. 12.Supernatant Removal: 1. Aspirate all supernatant, taking care as the pellet may not adhere tightly to the tube. 13.Ethanol Evaporation: 1. Remove any ethanol beads and let the open tube stand at room temperature until ethanol evaporates (5-10 minutes). 14.Storage: 1. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A. 2. Gently vortex the solution and store the DNA at -20°C.  Boiling Method for Plasmid DNA Preparation Principles: The boiling method is a classic alternative to the alkaline lysis method for plasmid DNA preparation. It follows the same principles as alkaline lysis. Materials 1- STET: 5% Triton X-100, 50mM Tris-Hcl, pH 8.0, 50 mM EDTA, pH 8.0, 8% sucrose. 2- Lysozyme: dry powder store at - 20 C. 3- Isopropanol 4- 70% ethanol. 5- TE: 10 mM Tris-Hcl, pH 7.5, 1 mM EDTA. 6- A boiling water bath. Methods for Plasmid DNA Extraction Using Boiling Method: noculation and Culture Setup: Inoculate 2-3 mL of broth containing an appropriate antibiotic with a bacterial colony. Incubate at 37°C overnight to allow bacterial growth. Bacterial Cell Harvest: Harvest bacterial cells by centrifugation for 2 minutes at 10,000 rpm. Remove the supernatant, leaving the bacterial pellet. STET Addition and Resuspension: Add 200 µL of STET to each tube containing the bacterial pellet. Resuspend the cells by vortexing. Boiling Step: Immediately place the tubes in boiling water for exactly 45 seconds.  Centrifugation and Pellet Formation: Centrifuge the tubes at 10,000 rpm for 10 minutes. Form a large, sticky, and loose pellet. Pellet Removal and Isopropanol Addition: Remove the pellet from each tube using a wooden toothpick. Add 200 µL of isopropanol to each tube. Centrifuge at 10,000 rpm for 5 minutes. Supernatant Aspiration and Ethanol Wash: Aspirate off the supernatant. Wash the pellet in 500 µL of 70% ethanol. Centrifuge for 1 minute to compact the pellet. Pellet Drying and Resuspension: Air dry the pellet for 10 minutes. Resuspend each pellet in 100 µL of TE buffer. Thank you