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Optimization of mass extraction conditions of plasmid DNA in Escherichia coli

Date:2021/01/06 13:44:40
Plasmids are closed circular DNA molecules that exist on the chromosomes of bacteria, yeasts and actinomycetes and express the genetic information they carry. They are often used as vectors in genetic engineering. Insert the target gene fragment into the plasmid to form a recombinant plasmid or recombination body. Then, the reconstituted recombinant plasmid is treated and transformed by electric shock, CaCl2 and other chemical reagents, and then transferred to the recipient bacteria (such as Escherichia coli) to make the target gene Reproduce or express in the recipient bacteria. By optimizing the steps in the kit extraction of plasmids, after DNA concentration measurement, it was found that the quality and purity of the plasmid DNA extracted by the optimized steps were better than those of the control group, and could better meet the requirements of subsequent experiments.
1Materials and methods
1.1 Material
1.1.1 Strains and plasmids
Recombinant plasmids pVAXl-HA2-FimA/IL-15 and pVAXl-HA2/IL-15 were constructed and preserved in the early stage of the class, (Escheriehia c0li, E.c0li) JMl09 was provided by the Key Laboratory of Stomatology of Zunyi Medical College.
1.1.2 Main reagents
The gold medal super endotoxin-free plasmid kit was purchased from Kangwei Century Biotechnology Co., Ltd.; kanamycin was purchased from Beijing Soleibao Company; DNAMarkerDL2,000 was purchased from Dalian TakaRa Bioengineering Company; DNAMarker 5000bp was purchased from Beijing biomed the company.
1.2 method
1.2.1 Extraction of plasmid DNA
The control group was carried out according to the kit steps of Kangwei Century Biotechnology Co., Ltd.
The experimental group is optimized based on the kit steps as follows:
(1) Pick a single colony in LB liquid medium with kanamycin, shake the bacteria overnight at 37°C, until the 0D600 value is 0.6. Take 150ml of the bacterial solution cultured overnight, add it to the centrifuge tube, centrifuge at 12000Xg for 3 minutes to collect the bacteria.
(2) Add 12ml of Buffer P1 to the centrifuge tube containing the bacterial pellet, and use a pipette or vortex to mix well to suspend the bacterial pellet.
(3) Add 12ml BufferP2 to the centrifuge tube, gently mix upside down and mix 10 times to fully lyse the bacteria, and leave it at room temperature for 5 minutes.
(4) Add 12ml BufferE3 to the centrifuge tube, and immediately mix upside down 10 times to avoid local precipitation. A white flocculent precipitate appeared at this time, and it was left at room temperature for 5 minutes. Centrifuge at 12000Xg for 10 minutes. After removing most of the precipitate with a pipette, pour all the supernatant into the endotoxin-removing filter, slowly push the handle to filter, and collect the filtrate in a clean 50ml centrifuge tube.
(5) Add 0.3 times the filtrate volume of isopropanol and 0.1 times the filtrate volume of sodium acetate to the filtrate, mix up and down.
(6) Add 2ml BufferPs to the treated adsorption column that has been loaded into the collection tube, centrifuge at 12000xg for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
(7) Transfer the mixed solution of the filtrate, isopropanol and sodium acetate in step 5 to a well-balanced adsorption column.
(8) Centrifuge at 12000Xg for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
(9) Add 10ml BufferPW to the adsorption column, centrifuge at 12000xg for 2 minutes, discard the waste liquid in the collection tube.
(10) Repeat step 9.
(11) Put the adsorption column back into the collection tube, centrifuge at 12000Xg for 5 minutes, centrifuge the liquid on the adsorption membrane clean, empty it once, discard the waste liquid, and place the adsorption column at room temperature for 10 minutes.
(12) Place the adsorption column in a new centrifuge tube, add 1ml Endo-freeBufferEB to the middle of the adsorption membrane, leave it at room temperature for 10 minutes, centrifuge at 12000Xg for 5 minutes, collect the plasmid solution in the centrifuge tube, and repeat the centrifugation twice. Store the plasmid at -20°C.
1. In order to increase the efficiency of plasmid recovery, the obtained solution can be newly added to the adsorption column, placed at room temperature for 5 minutes at 12000Xg and centrifuged for 5 minutes, and the plasmid solution is collected in a centrifuge tube. 2. When the number of plasmids is low or >10kb, Endo-fleeBufferEB is preheated in a 65-70°C water bath to increase the extraction efficiency. 1.2.2 Plasmid DNA concentration detection
The concentration of Escherichia coli plasmid DNA extracted from the experimental group and the control group was detected by an ultra-micro nucleic acid protein analyzer. 1.2.3 Enzyme digestion experiment of plasmid DNA
Recombinant plasmids pVAXl-HA2-FimA and pVAXl-HA2-FimA/IL-15 were digested with Xho I/Nhe I, 1% agarose gel electrophoresis 100v, 1h, took pictures with the automatic gel imaging analysis system, and recorded the results. 2 results
For the bacteria in the same culture medium, the plasmids were extracted by the above-mentioned different methods, and the plasmid concentration was detected by the ultra-micro nucleic acid protein analyzer. The results clearly showed that the plasmids extracted by the optimization step were relatively pure and the concentration was high (see table 1). Plasmid digestion identification: 2.2 kb (pVAXl-HA2-FimA, Figure 1), 3.2 kb (pVAXl-HA2-FimA/IL-15, Figure 2) gene fragments can be seen. The digestion effect is very ideal. The belt size is the same, and there is no miscellaneous belt, which is the same as expected. 3 Discussion 
Plasmid extraction is the basis of genetic engineering. All methods of extracting plasmid DNA basically include three steps: culturing bacteria to amplify the plasmid; collecting and lysing bacteria; and isolating and purifying plasmid DNA. At present, the methods of plasmid extraction mainly include alkaline lysis method, boiling method, toothpick method, etc. Among them, alkaline lysis method is widely used. There are differences in denaturation and renaturation between chromosomal DNA and plasmid DNA. The alkaline lysis method is based on the separation of plasmid DNA. Plasmid extraction kit is based on the alkaline lysis method to lyse cells. It uses a unique silicon matrix membrane adsorption technology to efficiently and exclusively bind plasmid DNA. At the same time, a special buffer system and endotoxin removal filter are used to effectively remove internal Toxins, genomic DNA, RNA, protein and other impurities have good extraction results. This experiment optimizes the plasmid extraction steps in the kit to obtain a higher efficiency plasmid extraction rate, which is beneficial to reduce experimental costs.

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