Plasmid preparation

Summary

Plasmid preparation can be used for (1) carrying exogenous genes into bacteria for amplification or expression of the main vector, which has an important role in gene manipulation; (2) isolation and extraction of plasmids is the most commonly used and basic experimental techniques.

Operation method

constructing a carrier

Principle

At pH 12.0 ~ 12.6 in an alkaline environment, the linear large molecular weight chromosomal DNA of bacteria denatured and separated, whereas the covalently closed-loop plasmid DNA was denatured but remained in a topologically entangled state. By adjusting the pH to neutral and in the presence of high salt and at low temperatures, most of the chromosomal DNA, large molecular weight RNA and proteins form a precipitate in the presence of the detergent SDS, while the plasmid DNA remains in a soluble state. By centrifugation, most of the cellular debris, chromosomal DNA, RNA and proteins can be removed, and the plasmid DNA is still in the supernatant, which is then extracted with phenol and chloroform to further purify the plasmid DNA.

Materials and Instruments

Escherichia coli bacteriophage
LA medium LB medium 75% ethanol
Shaker Centrifuge Ultra-clean table TE

Move

1. Take E. coli bacterial fluid containing pUC18 plasmid and incubate it on LA medium at 37℃ overnight;

2. Pick single colonies with a sterile toothpick and inoculate them in LB medium containing Amp antibiotic and incubate them overnight at 37℃ on shaker - 250 r/min;

3. Aspirate 1.5 ml of bacterial solution, centrifuge at 12000 g for 2 min, collect the bacterial body and pour off the bacterial solution; aspirate 1.5 ml of bacterial solution, collect the bacterial body again and pour off the bacterial solution as much as possible;

4. Add 300 ml of solution I and shake well, re-suspend the cells and shake well (Note: the precipitate or debris should be thoroughly beaten well);

5. Add 300 ml of Solution II, mix gently by inverting, and leave until clear, usually no more than 5 minutes;

6. add 300 ml of Solution III, mix by inversion and leave on ice for 10 minutes to allow impurities to settle well;

7. Centrifuge at 12000 g for 10 minutes;

8. Aspirate 800 ml of supernatant (note: do not aspirate floating impurities) into another Eppendorf tube, add 2/3 volume of isopropanol, and leave at room temperature for 5 minutes;

9. Centrifuge at 12000 g for 15 minutes at room temperature;

10. Pour off the supernatant and wash with 75% ethanol to remove salt (leave for a few moments or centrifuge for 3 minutes and pour off the supernatant);

11. Leave the DNA at room temperature or air-dry on an ultra-clean bench;

12. Add 40 ml of sterilized ultrapure water or TE to dissolve;

13. Quality check of plasmid and BAC, and store at -20℃.

Caveat

A small amount of prepared plasmid DNA can be extracted by phenol/chloroform for restriction endonuclease analysis, but for some experiments with high DNA purification requirements, such as mammalian cell transfection and transgenic animal manipulation, the purity of plasmid DNA needs to be further improved. This purity requirement not only includes the removal of bacterial chromosomal DNA, RNA and protein, but also the selection of different molecular configurations of plasmid DNA, generally for the purification of covalent closed-loop plasmid DNA. Commonly used methods are as follows:①Polyethylene glycol precipitation; ②Cesium chloride - ethyl bromide② Cesium chloride - ethidium bromide density gradient centrifugation: for many years is still the basic method to provide milligrams of high-purity plasmid DNA, the method requires the use of ethidium bromide and a long time ultracentrifugation, it can prepare a large number of plasmid DNA, but more expensive and time-consuming.Sepharose C L-4B and Bio-Gel A-150 m chromatography.NaCl ultracentrifugation.

Common Problems

Note: Plasmid detection

Electrophoresis: plasmid electrophoresis generally has three bands, respectively, for the plasmid's superhelix, open loop, linear three configurations.

Absorbance value detection: using spectrophotometer to detect the absorbance value of 260nm, 280nm wavelength, if the ratio of absorbance value of 260nm/280nm is between 1.7-1.9, it indicates that the quality of plasmid is better, and 1.8 is the best, lower than 1.8 indicates that there is protein contamination, and greater than 1.8 indicates that there is RNA contamination.

Sourced from "Preparation of plasmids" Journal of Anhui Medical University.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/