Extraction of plasmid DNA by alkaline lysis

Purpose

1. Master the most commonly used methods and detection methods for plasmid DNA extraction;

2. Understand the preparation principle and the role of various reagents.

Experimental principle 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 the denaturation and renaturation of chromosomal DNA and plasmid DNA to achieve the purpose of separation. Under alkaline conditions with a pH value of up to 12.6, the hydrogen DNA bond of the chromosomal DNA is 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.

Bacterial plasmids are a type of double-stranded, closed-loop DNA, ranging in size from 1 kb to more than 200 kb. Various plasmids are self-replicating genetic components that exist in the cytoplasm and are independent of the cell chromosomes. Usually, they are continuously and stably in a free state outside the chromosome, but they can also be reversibly integrated into the host chromosome under certain conditions. On the other hand, it replicates with the replication of chromosomes and is transmitted to the offspring through cell division.

Plasmids have become the most commonly used carrier molecule for gene cloning. The important condition is that a large number of purified plasmid DNA molecules can be obtained. There are many methods available for the extraction of plasmid DNA. In this experiment, the alkaline lysis method was used to extract the plasmid DNA

1. Materials

Escherichia coli containing pUC19 plasmid, 1.5ml plastic centrifuge tube, centrifuge tube holder, pipette tip and box, toilet paper.

2. Equipment

Micropipette (20μl, 200μl, 1000μl), desktop high-speed centrifuge, constant temperature shaking shaker, high-pressure steam sterilizer (sterilizer), vortex oscillator, constant temperature water bath, double steamer, refrigerator, etc.

3. Reagent preparation

1. LB liquid culture medium: weigh 10g of peptone (Tryptone), 5g of yeast extract (Yeast extract), 10g of NaCl, dissolve in 800ml of distilled water, adjust pH to 7.5 with NaOH, add water to a total volume of 1 liter, steam under high pressure Sterilize for 15 minutes.

2. Ampicillin (Apicillin, Amp) mother liquor: formulated into 100mg / ml aqueous solution, stored at -20 ℃ until use.

3. Solution I: 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0).

Preparation method: 1M Tris-HCl (pH 8.0) 12.5ml, 0.5M EDTA (pH 8.0) 10ml, glucose 4.730g, add ddH2O to 500ml. Autoclave at 121 ° C for 15min and store at 4 ° C.

4. Solution II: 0.2M NaOH, 1% SDS.

Preparation method: 2M NaOH 1ml, 10% SDS 1ml, add ddH2O to 10ml. Temporary configuration before use.

5. Solution III: potassium acetate (KAc) buffer, pH 4.8.

Preparation method: 5M KAc 300ml, glacial acetic acid 57.5ml, add ddH2O to 500ml. Store at 4 ℃ for future use.

6. Phenol / chloroform / isoamyl alcohol (25: 24: 1). Chloroform denatures the protein and helps to separate the liquid phase from the organic phase, and isoamyl alcohol can eliminate foaming during the extraction process. Both phenol and chloroform are highly corrosive, and gloves should be worn during operation. 【Bio Show】

7. Absolute ethanol;

8. 70% ethanol;

9. TE: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0).

Preparation method: 1M Tris-HCl (pH 8.0) 1ml, 0.5M EDTA (pH 8.0) 0.2ml, add ddH2O to 100ml. Sterilize at 121 ℃ for 20min under high pressure and humidity, and store at 4 ℃ for future use.

1M Tris Cl (Tris (trishydroxymethyl) aminomethane): Dissolve 121g Tris base in 800ml H2O, adjust the pH value with concentrated hydrochloric acid, add water to 1L after mixing;

0.5MEDTA (ethylenediaminetetraacetic acid): Dissolve 186.1g Na2EDTA.2H2O in 700ml H2O, adjust the pH to 8.0 with 10M NaOH (requires about 50ml), make up H2O to 1L.

10. RNase A mother liquor: dissolve RNase A in 10mmol / LTris · Cl (pH7.5), 15mmol / L NaCl, make a 10mg / ml solution, and heat at 100 ℃ for 15 minutes to make the mixed DNA The enzyme is inactivated. After cooling, use a 1.5ml eppendorf tube to divide into small portions and store at -20 ℃.

4. Operation steps

1. Pick single colony grown on LB solid medium, inoculate in 20ml LB (containing Amp100ug / ml) liquid medium, and culture at 37 ℃, 250rmp shaking overnight (about 12-14hr).

2. Pour 1.5ml of culture solution into a 1.5ml eppendorf tube and centrifuge at 12000rmp for 1-2min. Discard the supernatant and put the centrifuge tube upside down on toilet paper to make the liquid drain as much as possible.

3. The bacterial pellet is resuspended in 100 μl of solution I (need to shake vigorously to make the bacterial cells evenly mixed.), And leave at room temperature for 5-10 min.

4. Add 200μl of freshly prepared solution II, close the mouth tightly, quickly and gently invert the eppendorf tube several times to mix the contents (do not shake), and ice bath for 5 min to lyse the cell membrane (Solution II is the lysate, The bacterial solution in the centrifuge tube gradually becomes clear).

5. Add 150μl of pre-chilled solution III, close the tube tightly, and invert the tube gently for several times to mix well, see white flocculent precipitate, which can be placed on ice for 5min. Centrifuge at 12000rmp for 10min. Solution III is a neutralizing solution. At this time, the plasmid DNA is renatured, and the chromosome and protein are irreversible, forming an insoluble complex, and at the same time, K + precipitates the SDS-protein complex.

6. Transfer the supernatant into a clean eppendorf tube, add an equal volume of phenol / chloroform / isoamyl alcohol, mix by shaking, and centrifuge at 12000 rpm for 10 minutes. (450μl of phenol / chloroform / isoamyl alcohol.)

7. Carefully remove the supernatant in a new microcentrifuge tube, add twice the volume of pre-cooled absolute ethanol, mix well, place at room temperature for 2-5min, and centrifuge at 12000rmp × 10min.

8. Discard the supernatant, put the tube opening upside down on toilet paper to make all the liquid flow out, add 1ml 70% ethanol to wash the precipitate once, and centrifuge at 12000rmp for 5 min.

9. Aspirate the supernatant, put the tube upside down on toilet paper to drain the liquid, and dry at room temperature.

10. Dissolve the precipitate in 20 μl TE buffer (pH 8.0, containing 20 μg / ml RnaseA, about 4 μl), 37 ° C water bath for 30 min to degrade RNA molecules, and store in a -20 ° C refrigerator.

Five experimental results:

Precautions

1. Keep the temperature as low as possible during the extraction process.

2. Precipitated DNA usually uses ice ethanol, and it can be completely precipitated by leaving it at a low temperature for a long time. Isopropanol can also be used to precipitate DNA (usually equal volume), and the precipitation is complete and fast, but the salt is often precipitated, so most of them use ethanol.

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