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Process for preparing plasmid in large scale with high yield

A large-scale, high-yield technology, applied in DNA preparation, sugar derivatives, organic chemistry, etc., can solve the problems of poor quality and purity, not suitable for gene therapy and gene immunity, etc., and achieve the effect of increasing yield

Inactive Publication Date: 2006-05-24
MILITARY VETERINARY INST MILITARY SUPPIES PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its quality and purity are not as good as the plasmids prepared by alkali denaturation-CsCl density gradient ultracentrifugation and column chromatography kits, and the amount of bacterial endotoxin (LPS) contained is too high, so it is not suitable for gene therapy and genetic immunity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] 1. Cultivation and collection of recombinant bacteria: Pick a single colony of interest and inoculate 55ml of LB medium (add corresponding amount of resistant drugs, the same below) to OD 600 ≈0.6, → Take 50ml (another 5ml for identification) and pour it into 1000ml LB medium for about 12-16 hours; → Centrifuge at 5000rpm for 10 minutes, discard the supernatant, and collect the bacteria.

[0015] 2. Protoplast preparation: add 50ml containing 20% ​​sucrose, 50mM Tris-HCl (pH8.0) to the bacterial precipitation, blow and beat to suspend; → ice bath for 3-5 minutes; → add 10ml with pre-cooled 0.25M Tris-HCl (pH8.0) .0) prepared fresh lysozyme solution (5mg / ml), ice bath for 5 minutes; → add 20ml pre-cooled 0.25M EDTA (pH8.0), ice bath for 5 minutes; → add 20ml pre-cooled 0.25M Tris- HCl (pH8.0), 37 ° C water bath for 5 minutes; → immediately 4 ° C, 6000 rpm, 10 minutes, discard the supernatant. (Ice bath conditions and exposure time are critical)

[0016] 3. Extraction a...

Embodiment 2

[0022] Purify pcDNA3 plasmid from BL21(DE3) transformed recombinant bacteria, 2000LB medium, 46mg plasmid.

[0023] 1. Colony culture and collection: Pick a single colony transformed with pcDNA3 and inoculate 110ml LB medium (add ampicillin to 60μg / ml) to OD 600 ≈0.6, → take 100ml and pour it into 2000ml LB medium and cultivate for about 12-16 hours;

[0024] → Centrifuge at 5000rpm for 10 minutes, discard the supernatant, and collect the bacteria.

[0025] 2. Protoplast preparation: add 100ml containing 20% ​​sucrose and 50mM Tris-HCl (pH8.0) to the bacterial pellet, and pipette to suspend;

[0026] → Ice bath for 5 minutes;

[0027] →Add 20ml of fresh lysozyme solution (5mg / ml) prepared with pre-cooled 0.25M Tris-HCl (pH8.0), and ice bath for 5 minutes;

[0028] → Add 40ml of pre-cooled 0.25M EDTA (pH8.0), ice bath for 5 minutes;

[0029] → Add 40ml of pre-cooled 0.25M Tris-HCl (pH8.0), bathe in water at 37°C for 5 minutes;

[0030] → Immediately at 4°C, 6000rpm, 10 min...

Embodiment 3

[0050] Purify pVAX1 plasmid from JM109 transformed recombinant bacteria, 1000LB medium, 21mg plasmid

[0051] 1. Colony culture and collection: Pick a single colony of JM109 transformed by pVAX1 and inoculate 55ml LB medium (kanamycin to 30μg / ml) to OD 600 ≈0.6, → take 50ml (the other 5ml is used for identification) and pour it into 1000ml LB medium for about 14 hours;

[0052] → Centrifuge at 5000rpm for 10 minutes, discard the supernatant, and collect the bacteria.

[0053] 2. Protoplast preparation: add 50ml containing 20% ​​sucrose and 50mM Tris-HCl (pH8.0) to the bacterial pellet, and pipette to suspend;

[0054] → Ice bath for 5 minutes;

[0055] →Add 10ml of fresh lysozyme solution (5mg / ml) prepared with pre-cooled 0.25M Tris-HCl (pH8.0), and ice bath for 5 minutes;

[0056] → Add 20ml of pre-cooled 0.25M EDTA (pH8.0), ice bath for 5 minutes;

[0057] → Add 20ml of pre-cooled 0.25M Tris-HCl (pH8.0), bathe in water at 37°C for 5 minutes;

[0058] → Immediately at 4°...

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Abstract

A high-yield large-scale plasmid preparation method of the present invention includes colony cultivation and collection: protoplast preparation: plasmid extraction and purification: DNA detector is used to measure the content of the plasmid and an appropriate enzyme is used for enzymatic identification; the process is compatible with alkali denaturation- The purity of the plasmid prepared by the CsCl density gradient ultracentrifugation method is equivalent, but the yield is greatly increased, and the plasmid yield per 1000ml of E. coli culture reaches 15-23mg. First pellet the plasmid in the supernatant using 20% ​​PEG 8000 Instead of using isopropanol, add the precipitate to 18TE solution to dissolve, and add proteinase K with a final concentration of 50-100ug / ml to digest. Its endotoxin content is extremely low, which is equivalent to that of alkali denaturation-CsCl density gradient ultracentrifugation.

Description

Technical field: [0001] The invention provides a high-yield large-scale plasmid preparation method, relates to the improvement of the alkali denaturation-PEG precipitation method, and belongs to the technical fields of molecular biology, gene therapy and gene immunity. Background technique: [0002] The classic methods for large-scale preparation of plasmids are alkali denaturation-CsCl density gradient ultracentrifugation and alkali denaturation-PEG precipitation. Alkaline denaturation-CsCl density gradient ultracentrifugation can produce high-quality plasmids, but it requires high equipment conditions, high cost, and low yield, so it is rarely used. The yield of the plasmid prepared by the alkali denaturation-PEG precipitation method is relatively large, and the yield per 1000ml of bacteria is about 4-10mg, which is much higher than the yield of the plasmid prepared by the alkali denaturation-CsCl density gradient ultracentrifugation method and column chromatography kit. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N1/20C07H21/04
Inventor 涂长春余兴龙张茂林
Owner MILITARY VETERINARY INST MILITARY SUPPIES PLA
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