A plasmid is an extrachromosomal DNA molecule with a double-stranded closed-loop structure. The plasmid has the ability to replicate autonomously, enabling progeny cells to maintain their constant copy number and express the genetic information they carry. At present, plasmids have been widely used as carriers for vectors of genes in genetic engineering. Extraction of plasmid DNA from E. coli is also the most basic method of molecular biology. The extraction of plasmid DNA is based on the fact that the plasmid DNA molecule is smaller than the chromosomal DNA and has a supercoiled covalently closed loop, thereby separating the plasmid DNA from the E. coli chromosomal DNA. The plasmid DNA extraction methods are as follows: alkali denaturation method, ethidium bromide-ruthenium chloride density gradient method, DNA plasmid release method, hydroxyapatite column layer method and acid phenol method.

1. Extraction of plasmid DNA by alkaline denaturing

【Fundamental】

The commonly used alkali denaturation method has the advantages of simple operation, rapidity, and high yield. The main principle is to use the difference between denaturation and renaturation of chromosomal DNA and plasmid DNA to achieve separation. Under alkaline denaturing conditions (pH12.6), the hydrogen bond of the chromosomal DNA is broken, the double helix structure is unfolded and denatured, and the hydrogen bond of the plasmid DNA is mostly broken, and the double helix is ​​partially unfolded, but the covalently closed cyclic structure is The two complementary strands are not completely separated. When the pH is adjusted to neutral with sodium acetate at pH 4.8, the denatured plasmid DNA returns to its original configuration, and the chromosomal DNA cannot be renatured, forming a dense dense network. Structure, after centrifugation, chromosomal DNA is precipitated together with macromolecular RNA, protein SDS complex, etc. due to different buoyant densities. The method of isolating plasmid DNA generally comprises three basic steps: culturing the bacteria to amplify the plasmid; collecting and lysing the bacteria; isolating and purifying the plasmid DNA.

【equipment】

1.1.5ml Eppendorf tube

2. Micro sampler

3. Petri dish

4. Benchtop high speed centrifuge

5. Autoclave

【Reagents】

All reagents must be autoclaved (except organic solvents)

1. Solution I 50mmol / L glucose

25mmol/L Tris-Cl (pH 8.0)

10mmol/L EDTA (pH 8.0)

2. Solution II 0.2mol/L NaOH

1% SDS

Prepare before use

3. Solution III 5mol / L potassium acetate 60ml

3mol/L glacial acetic acid 11.5ml

ddH2O 28.5ml

pH 5.2

4.RNase A: 10mg/ml

5.TE buffer: 1mmol/L EDTA in 10mmol/L Tris-Cl (pH 8.0)

6.Tris-Cl (pH 8.0) saturated phenol

7. Chloroform/isoamyl alcohol (v/v): 24/1

8.70%: ethanol

9.LB/Amp: LB/ampicillin (50μg/ml)

【Steps】

1. Remove one colony from the selective culture plate and transfer to a tube containing 2 ml of LB/Amp medium. Incubate at 37 ° C for 12-16 h with shaking.

2. Transfer 1.5 ml culture to 1.5 ml Eppendorf tubes and centrifuge at 4000 rpm for 8 min.

3. Discard the supernatant, suspend the bacterial pellet in 100 μl of pre-cooled solution I, and add 2.5 μl of RNase A (10 mg/ml).

4. Add 200 μl of freshly prepared solution II to cap the tube. Quickly invert the tube 5 times to mix the contents and place on ice for 5 min.

5. Add 150 μl of pre-cooled solution III to cap the tube. The tube was inverted 5-8 times and placed on ice for 20 min.

6. Centrifuge (1000 rpm, 4 ° C, 5 min) and transfer the supernatant to another centrifuge tube.

7. Add an equal volume of benzene/chloroform/isoamyl alcohol, mix by shaking, and centrifuge as above.

8. Transfer the aqueous phase to another centrifuge tube, add 2.5 volumes of absolute ethanol, mix and place on ice for 20 min.

Centrifuge at 9.10 000 rpm for 4 min at 4 °C.

10. Discard the supernatant, wash the DNA pellet by adding 0.5 ml of 70% ethanol, discard the supernatant, and dry the plasmid DNA in air for 10 min.

11. The plasmid DNA was dissolved in 20 μl of ddH2O and stored at 4 °C.

2. Plasmid DNA miniprep kit for extraction of plasmid DNA

【Fundamental】

This kit is used for small-scale purification of plasmid DNA. The kit uses the traditional SDS alkaline lysis method combined with DNA preparation membrane technology, which is characterized by high efficiency, fastness and convenience. The complete operation can be completed in one hour. Using this kit, 1 to 20 μg of high-purity plasmid DNA can be purified from 1-4 ml of the overnight culture solution, and the plasmid DNA can be directly used for DNA sequence analysis and various enzymatic reactions.

【equipment】

Centrifuge

2. Spin Column

3. Collection Tube (2ml)

【Reagents】

1.RNase A1 (50mg/ml)*1 6μl

2.Solution I*1 3ml

3.Solution II*2 3ml

4.Solution III 4.8ml

5.Rinse A 5.5ml

6.Rinse B 16ml

7.Elution Buffer*3 0.8ml

*1 When using the kit for the first time, add all RNase A1 to SolutionI, mix well and store at 4 °C.

*2 If precipitation occurs, please dissolve at 37 ° C, and use it after returning to room temperature.

*3 Elution Buffer composition: 2.5 mmol/L Tris-Cl (pH 8.5).

[Operation method]

1. Cultivation of E. coli.

Single colonies were picked from the plate medium and inoculated into 1 to 4 ml of a liquid medium containing antibiotics, and cultured overnight at 37 °C. Note) The culture solution should not be excessive. When the culture solution is excessive, the amount of bacteria will be too large and the lysis will be insufficient. The purity of the plasmid will be affected during purification.

2. Take 1 to 4 ml of the overnight culture solution, centrifuge at 12 O0 rpm for 2 min, and discard the supernatant.

3. Fully suspend the bacterial pellet with 250 μl of Solution I (containing RNase A1). Note) Be careful not to leave small bacteria, and use a vibrating device such as a vibrator (Vortex) to fully suspend the cells.

4. Add 250 μl of Solution II and gently mix it upside down 5 to 6 times to fully lyse the cells to form a clear solution. Note) This step should not exceed 5 minutes.

5. Add 400 μl of 4 ° C pre-cooled Solution III, gently mix upside down 5 to 6 times until a compact agglomerate is formed, then let stand at room temperature for 2 min.

6. Centrifuge at 12 000 rpm for 5 min at room temperature and take the supernatant. Note) At this time, centrifugation at 4 °C is not conducive to sedimentation.

7. Place the Spin Column in the kit on the Collection Tube.

8. Transfer the supernatant of the above operation 6 to the Spin Column and centrifuge at 360 °C for 1 min (if there is liquid residue in the SpinColumn, the centrifugal speed can be increased appropriately, and then centrifuged for 1 min), and the filtrate is discarded.

9. Add 500 μl of Rinse A to the Spin Column, centrifuge 3OSec at 3600 rpm, and discard the filtrate.

10. Add 700 μl of Rinse B to the SPin Column, centrifuge 3 OSec at 360 ° rpm, and discard the filtrate.

11. Repeat step 10 and centrifuge for 1 min at 12000 rpm.

12. Place the Spin Column on a new 1.5 ml centrifuge tube and add 60 μl of water or eluate to the center of the Spin Column membrane and let stand for 1 min at room temperature. Note) When the water or eluent is heated to 60 ° C, it is beneficial to improve the elution efficiency.

13.12 O00 rpm Centrifuge for 1 min to collect DNA.

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