Plasmid DNA extraction experiment
Plasmid DNA extraction can: (1) rapid purification of plasmids; (2) for molecular biology experiments such as sequencing, in vitro transcription and translation, restriction enzyme digestion, bacterial transformation, etc. Principle of the experimental method Alkaline lysis method is the most widely used method for preparing plasmid DNA. Alkaline denaturation extraction of plasmid DNA is based on the difference between denaturation and renaturation of chromosomal DNA and plasmid DNA for separation purposes. Under alkaline conditions with a pH of up to 12.6, the hydrogen bonds of the chromosomal DNA are broken, and the double helix structure is unwound and denatured. Most of the hydrogen bonds of the plasmid DNA are also broken, but the two complementary strands of the supercoiled covalently closed loop will not be completely separated. When the pH value is adjusted to neutral with NaAc / KAc high salt buffer pH 4.8 The denatured plasmid DNA restores its original configuration and is stored in solution, while the chromosomal DNA cannot refold and form a tangled network structure. Through centrifugation, the chromosomal DNA and the unstable macromolecular RNA, protein-SDS complex Wait for it to settle down and be removed. Concise principle: The alkaline lysis method is based on the difference between denaturation and renaturation of DNA to achieve the purpose of separation. Alkaline denatures the plasmid DNA, and then adjusts the pH to neutral to renature. The renature is the plasmid DNA, but the chromosomal DNA does not renature, and tangles into a net-like substance, which is removed by centrifugation.
Experimental material: bacterial liquid
Reagents and kits: plasmid extraction kit RNase A Buffer S1 Buffer S2 Buffer S3 Buffer W1 Buffer W2 concentrate Eluent Instruments, consumables: DNA preparation tubes centrifuge tubes pipette gun tips pipette tip box centrifuges
Experimental step 1. Kit composition, storage, stability consumables: DNA preparation tube, 2 ml centrifuge tube, 1.5 ml centrifuge tube. RNase A: 50 mg / ml, stored at room temperature. Buffer S1: bacterial suspension. After adding RNase A, store at 4 ° C. Buffer S2: Bacterial lysate, store in airtight at room temperature. (If precipitation occurs, it should be dissolved in a 37 ° C warm bath and cooled to room temperature before use.) Buffer S3: Neutralizing solution, store at room temperature in a closed place. Buffer W1: Washing solution, store in airtight at room temperature. Buffer W2 concentrate: Desalting liquid. Before use, add ethanol according to the quantity on the bottle, mix well, and store at room temperature in a sealed place. Available 100% ethanol or 95% ethanol. Eluent: 2.5 mM Tris-HCl, pH 8.5, sealed at room temperature.
2. Experimental preparation 1. Before the first use, add RNase A to Buffer S1 and store at 4 ° C. 2. Prepare Tip and centrifuge tubes free of nucleic acid and nuclease contamination. 3. Before the first use, add a specified volume of absolute ethanol to Buffer W2 concentrate.
3. Operation steps 1. Take 1-4 ml of bacterial solution cultured in LB medium overnight (if rich medium is used, the volume of bacterial solution should be halved or less), centrifuge at 12 000 × g for 1 min, and discard clear. 2. Suspend the bacterial pellet with 250 μl of Buffer S1 to which RNase A has been added. Suspension needs to be even, and no small bacterial lumps should be left. 3. Add 250 μl of Buffer S2, gently and fully upside down and mix 4-6 times to evenly lyse the cells until a translucent solution is formed. This step should not exceed 5 min. 4. Add 350 μl Buffer S3, mix gently and upside down 6-8 times, and centrifuge at 12 000 × g for 10 min. Steps 5 to 7 can choose negative pressure method or centrifugation method to purify plasmid DNA. A. Negative pressure method 5A. Insert the plasmid DNA preparation tube into the interface of the negative pressure device. Pipette the centrifuged supernatant from step 4 into a preparation tube, turn on and adjust the negative pressure to -20-30 inches of mercury, and slowly aspirate the solution from the tube. 6A. Add 500 μl Buffer W1 and aspirate the medium solution. 7A. Add 700 μl Buffer W2 and aspirate the medium solution; wash again with 700 μl Buffer W2 in the same way. * Make sure to add absolute ethanol to the specified volume on the reagent bottle in Buffer W2 concentrate. B. Centrifugation method 5B. Pipette the centrifuged supernatant from step 4 and transfer to a DNA preparation tube (placed in a 2 ml centrifuge tube) and centrifuge at 12,000 × g for 1 min. 6B. Put the preparation tube back into the centrifuge tube, add 500 μl Buffer W1, centrifuge at 12,000 × g for 1 min, and discard the filtrate. 7B. Put the preparation tube back into the centrifuge tube, add 700 μl Buffer W2, centrifuge at 12,000 × g for 1 min, and discard the filtrate; wash again with 700 μl Buffer W2 in the same way. Discard the filtrate. * Make sure to add absolute ethanol to the specified volume on the reagent bottle in Buffer W2 concentrate. 8. Put the preparation tube back into a 2 ml centrifuge tube and centrifuge at 12,000 × g for 1 min. 9. Move the preparation tube into a new 1.5 ml centrifuge tube, add 60-80 μl of Water or Eluent to the center of the DNA preparation membrane, and let stand at room temperature for 1 min. Centrifuge at 12,000 × g for 1 min.
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