DNA recombination in vitro

Summary

DNA recombination, also known as gene cloning, is the most basic technique in gene manipulation and the core technology of molecular biology.DNA recombination refers to the application of enzymology to synthesize DNA molecules with self-replicating ability by joining genetic material from various sources (homologous or heterologous, prokaryotic or eukaryotic, natural or artificial) with vector DNA to form replicons (DNA molecules) in vitro, followed by transformation or transfection of host cells to screen the cells containing the target gene, and then amplification and extraction to obtain a large number of the same DNA molecule. After that, the host cells are transformed or transfected to screen out the transformant cells containing the target gene, and then amplified and extracted to obtain a large number of the same DNA molecule, and the basic process is illustrated as follows:

Principle

The basic principle of DNA recombination in vitro is the process of forming a new DNA molecule by joining two or more DNA molecules in vitro and cloning and amplifying them in vivo.

Appliance

DNA recombination in vitro is an important intermediate process for the construction of gene libraries, exogenous expression of genes, and gene modification, i.e., targeted mutagenesis of genes.

Operation method

DNA recombination in vitro

Principle

The basic principle of DNA recombination in vitro is the process of forming a new DNA molecule by joining two or more DNA molecules in vitro and cloning and amplifying them in vivo.

Materials and Instruments

Reagents:
Restriction endonuclease, DNA ligase, vectors, ethanol, chloroform/isoamyl alcohol, RNase, agarose, ethidium bromide, E. coli strain, LB medium, 100 mM CaCl
2
DMSO/
2
-mercaptoethanol
Instruments:
Water bath, PCR instrument, incubator, centrifuge, oscillator

Move

The basic steps of DNA recombination in vitro include: I. Preparation of genomic DNA(a) Extraction of genomic DNA from mammalian tissues

1. Cut fresh tissues into small pieces and immediately freeze them in liquid nitrogen;

2. Crush 200 mg~1 g of tissue with a pre-cooled mortar or pound it into fine powder with a hammer, suspend every 100 mg of tissue with 1.2 ml of digestion buffer, and then incubate at 50℃ for 12~18 h with shaking;

3. Extract the sample with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuge at 1700×g for 10 min. If the sample does not dissolve well, add 1 volume of digestion buffer without proteinase K and repeat the centrifugation. If there is a thick white layer at the interface, repeat the extraction and transfer the upper layer (aqueous solution) to a new centrifuge tube;

4. Add 1/2 volume of 7.5mol/L ammonium acetate and 2 times volume of 100% ethanol and centrifuge at 12,000 rpm for 2 min;

5. Wash with 70% ethanol, air dry, and re-dissolve the precipitate in TE buffer or sterile distilled water to a final concentration of about 1 mg/ml. Note: Add 0.1% SDS and 1?ng/ml of RNAase without DNAzyme, and incubate at 37°C for 1 h to remove residual RNA. Repeat Steps 4 and 5. 1 g of the solution should be added.

6. Repeat steps 4 and 5. 2 mg of DNA can be extracted from 1 g of cells.

(ii) Extraction of genomic DNA from plant tissues

1. Add 2-mercaptoethanol to the desired amount of CTAB extraction solution to achieve a final concentration of 2% (v/v). Heat this solution as well as the CTAB/NaCl solution to 65 °C. Approximately 4 ml of 2-mercaptoethanol/CTAB Extract and 0.4 to 0.5 ml of CTAB/NaCl Solution are required for each gram of fresh leaf tissue;

2. Using a liquid nitrogen (- 196°C) or dry ice (- 78°C) cryo-homogenizer, pulverize the plant tissues to a fine powder and transfer the frozen tissues to a test tube or flask containing an organic solvent;

3. Add preheated 2-ME/CTAB to the pulverized tissues, mix to moisten thoroughly, and incubate at 65°C for 10-60 min with occasional mixing;

4. The homogenate was extracted with an equal volume of 24:1 chloroform/isoamyl alcohol, mixed thoroughly by turning up and down, and centrifuged at 7500×g for 5 min at 4°C. The upper aqueous phase was recovered;

5. Add 1/10 times the volume of 65 ℃ preheated CTAB/NaCl solution, upside down and mix well;

6. Extract with equal volume of chloroform/isoamyl alcohol, mix well, centrifuge, recover the upper aqueous phase;

7. Add 1 times the volume of CTAB precipitation solution, mix upside down, if precipitation is visible, continue to step 8, or incubate at 65 ℃ for 30 min;

8. Centrifuge at 500×g, 4°C (or ~2700rpm) for 5 min;

9. Remove the supernatant and resuspend the precipitate in high-salt TE buffer (0.5-1 ml per gram of starting material), if the precipitate is difficult to dissolve, incubate at 65°C until all or most of the precipitate is dissolved;

10. Add 0.6 times the volume of isopropanol to precipitate nucleic acids, mix thoroughly, and centrifuge at 7500×g for 15 min at 4℃;

11. Wash the precipitate with 75% ethanol, dry, and resuspend with as little TE or water as possible (0.1~0.5 ml per gram of starting material).

Preparation of plasmid DNA(a) Small volume preparation of plasmid.

1. Add 5 ml of LB culture medium (containing screening antibiotics) to a 15 ml well ventilated test tube, then inoculate a single colony of the transformed bacteria into the culture medium and incubate for 14-16 h at 37℃ with oscillation at a speed of 225 rpm;

2. The culture medium was then centrifuged at 10,000 rpm for 5 min at 4°C, and the culture medium was discarded;

3. Suspend the bacteria with pre-cooled cell suspension and leave for 5 min at room temperature;

4. Add Bacterial Lysis Solution, turn the microcentrifuge tube up and down to lyse the bacteria, and wait until the solution becomes viscous;
   

5. Add Neutralization Solution, mix upside down and place on ice for 5 min, centrifuge at 12,000 rpm for 5 min at 4℃;

6. Transfer the supernatant to another centrifuge tube (be careful not to mix in the white precipitate), add an equal volume of TE saturated phenol/chloroform (1:1) mixture, shake and mix for 1 min, centrifuge at 12,000 rpm for 2 min;

7. Transfer the upper aqueous phase to another centrifuge tube, add an equal volume of chloroform/isoamyl alcohol (24:1) solution, shake well for 1 min, and centrifuge at 12,000 rpm for 2 min;

8. Transfer the upper aqueous phase to another centrifuge tube, add 2.5 times the volume of anhydrous ethanol, and settle at -20°C for 15-30 min, then centrifuge at 12,000 rpm for 15 min;

9. Dispose of the supernatant, add 100 ng L of distilled water or TE (pH 8.0) to the precipitate, dissolve the precipitate, add 20 ng/ml RNase, and leave it at room temperature for 30 min, then add an equal volume of chloroform/isoamyl alcohol (24:1), shake and mix for 1 min, and then centrifuge at 12,000 rpm for 2 min, then transfer the upper aqueous phase to another centrifuge tube and add 0.1 times volume of 3M NaAc (pH 5.2). Transfer the upper aqueous phase to another centrifuge tube, add 0.1 times the volume of 3M NaAc (pH5.2) and 2.5 times the volume of anhydrous ethanol, and settle for 15-30 min at -20℃, then centrifuge for 15 min;

10. Discard the supernatant, add 70% ethanol (careful not to mix) and centrifuge at 12000rpm for 10 min;

11. Discard the supernatant and dry the plasmid DNA under vacuum or naturally;

12. Dissolve the plasmid DNA in deionized water and store at -20℃.

(ii) Mass preparation of plasmid.

1. Inoculate 5 ml of the transformed strain into 500 ml LB culture medium (containing antibiotics for screening), and incubate at 37℃ for 12~16 hours with shaking at 225rpm;

2. Collect the bacteria by centrifugation at 10,000 rpm for 15 min at 4℃;

3. Bacteria were suspended in 100 ml of pre-cooled STE (0.1 M NaCl, 10 mM Tris.Cl, pH 8.0, 1 mM EDTA, pH 8.0) and collected by centrifugation at 12,000 rpm;

4. Bacteria were suspended in 10 ml of bacterial suspension and transferred to a 50 ml centrifuge tube;

5. Add 1 ml of freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris.Cl (pH 8.0), mix and incubate for 30 min at room temperature;

6. Add 15 ml of freshly prepared cell lysate and mix upside down until the solution becomes viscous;

7. Add 10 ml of pre-cooled neutralization solution, mix upside down and ice bath for 10 min;

8. Centrifuge at 12,000 rpm at 4°C for 20 min;

9. The supernatant was filtered through four layers of sterilized gauze into another 50 ml centrifuge tube, 0.6 times the volume of isopropanol was added, and left at room temperature for 10 min, then centrifuged at 12,000 rpm for 5 min at room temperature;

10. The supernatant was discarded, and water and RNase were added to the precipitate, which was left at room temperature for 30 min. The supernatant was then extracted once with chloroform/isoamyl alcohol (24:1), and the upper aqueous phase was mixed with 0.1 times the volume of 3M NaAC (pH 5.2) and 2.5 times the volume of anhydrous ethanol, and then precipitated at -20℃ for 15 min, and centrifuged at 12,000 rpm for 15 min;

11. Discard the supernatant, wash with 75% ethanol at room temperature, centrifuge at 12,000 rpm for 10 min;

12. Discard the supernatant and dry the plasmid DNA under vacuum or naturally;

13. Add appropriate amount of deionized water or TE buffer (pH8.0) to dissolve the plasmid DNA, and store at -20℃.

Recovery of DNA fragments after enzymatic digestion and vector ligation

1. For the DNA digestion reaction, inactivate the enzyme activity by heating at 75℃ for 15 min;

2. After mixing 10×CIP buffer and 1U CIP, incubate at 37℃ for 30-60 min, then heat at 75℃ for 15 min to inactivate CIP activity;

3. Separate and purify the target DNA fragments for ligation reaction by agarose gel electrophoresis.

Ligation reaction: after mixing the ligation system, put the water bath at 14-16 ℃ for 6-12 h.

IV. Transformation of recombinant plasmids(a) Preparation of receptor cells

1. The E. coli strain in LB agar medium drawn lines, 37 ℃ culture 12 ~ 16 h. The next day from the agar plate on the line;

2. On the next day, take a single colony from the agar plate and put it into 2 ml of LB medium, and incubate it at 37℃ with 200 rpm for 12-16 h. Take 1 ml of the above culture and inoculate it into the LB medium, and incubate it at 37℃ with 200 rpm;

3. Take 1 ml of the above culture and inoculate into 100 ml LB medium, incubate at 37℃ with 200 rpm until the A600 value is 0.5～0.6 (about 3 h);

4. Put the bacterial solution in an ice bath for 10 min, and then centrifuge at 2,500×g for 20 min at 4℃ to collect the bacterial solution;

5. Discard the supernatant, invert the centrifuge tube for 1 min to remove the remaining liquid, and then add 10 ml of ice-cold 100 mM _{CaCl2 solution} to suspend the cells, and then centrifuge at 4000 rpm, 4℃ for 10 min;

6. Centrifuge at 4000rpm, 4℃ for 10 min to recover the cells, discard the supernatant, and add 2 ml of ice-cold 100 mM CaCl2 solution to every 50 ml of original culture to suspend the cells;

7. The cells can be stored at 4℃ for 1~2 weeks in 200 nil l portions. For long-term storage, add glycerol to a final concentration of 15% and store at -70℃.
   

(ii) Transformation of recombinant plasmids

1. Melt the receptor cells on ice, then add DMSO or 2-mercaptoethanol, mix and add L-conjugate (containing recombinant plasmid), mix gently, and place on ice for 30 min;

2. Add DMSO or 2-mercaptoethanol, mix and add L-conjugation reaction solution (containing recombinant plasmid), mix gently and put on ice for 30 min;

3. Add 2 ml of LB culture medium and incubate for 1 h at 37℃ with gentle shaking;

4. Centrifuge at 4,000×g for 10 seconds, discard the supernatant, and resuspend the organisms with LB culture medium;

5. Spread the bacterial solution on the LB agar culture plate containing appropriate antibiotics, spread it well, leave it at room temperature for 20-30 min, and incubate for 12-16 h in an incubator at 37℃.

(iii) Screening of transformed cells(D) Storage of plasmids and strains

1. The plasmid can be kept frozen at -20℃.

2. The strains can be stored in culture medium containing 8-15% glycerol at -20℃ or -70℃. They can also be stored in semi-solid LB agar medium by puncture.

Caveat

Precautions for DNA recombination in vitro are:(a) the basic principles of carrier selection: first, clear selection of the basic purpose of the carrier, and second, to understand the basic structure and functional components of the carrier. Carrier selection should generally consider the following points: 1 the size of the target DNA fragments; 2 clear recombination purposes; 3 suitable cloning sites; 4 the stability of the carrier.(b) Factors to be considered in the selection of vectors: In addition to the above basic principles, the selection of vectors should also take into account a number of factors that may have an impact on the manipulation of DNA, including some of the important components provided by the vector and their location, the sites that can be used for manipulation as well as the behavior of vectors in the host cell, and so on.(iii) Because RNA is susceptible to degradation by RNase; the whole operation should not be contaminated with RNase, and care should be taken to ensure aseptic operation.(iv) All reagents are prepared with DEPC-treated water.(e) Ethidium bromide is a carcinogen and moderately toxic, so wear waterproof gloves and do not pollute the environment. Protective glasses should be worn when observing the results under ultraviolet light.

Common Problems

Common problem of DNA recombination in vitro: low ligation efficiency;

Solution: Usually the molar ratio of DNA insertion fragment to vector DNA is 3:1 or 2:1, which needs to be calculated according to the molecular weight of DNA, not depending on the concentration; in the case of flush end ligation, the amount of insertion fragment should be much larger than that of vector DNA, which can be 5:1 or more. Sometimes the DNA has a sticky end and a flat end, which is similar to the double-enzyme cleavage sticky-end ligation; when mismatched ends need to be ligated, the ends are first processed to be flat, and then the ligation reaction is carried out.

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