The plasmid extraction method removes RNA, separates the plasmid from the bacterial genomic DNA, and removes proteins and other impurities to obtain a relatively pure plasmid. Today we mainly to understand the principle and operation steps of plasmid extraction.
1. Principle of plasmid extraction
Three solutions and a silicate fiber membrane (ultrafiltration column) were used. Solution I: 50 mM glucose / 25 mMTris-HCl / 10 mM EDTA, pH 8.0; Solution II: 0.2 N NaOH / 1% SDS; Solution III: 3 M potassium acetate / 2 M acetic acid / 75% alcohol.
Solution I
Glucose is such that E. coli after suspension does not deposit rapidly to the bottom of the tube; EDTA is a chelating agent for divalent metal ions such as Ca2+ and Mg2+, and its main purpose is to chelate divalent metal ions to inhibit the activity of DNase; Add RNase A to digest RNA.
Solution II
This step is alkali treatment. Among them, NaOH is mainly used to dissolve cells and release DNA, because in the case of strong alkalinity, the cell membrane undergoes a change from a bilayer membrane structure to a microcapsule structure. SDS is combined with NaOH in order to enhance the strong alkalinity of NaOH, while SDS acts as an anionic surfactant to destroy the lipid bilayer membrane. There are two things to keep in mind in this step: First, the time should not be too long, because the genomic DNA fragments will slowly break under such alkaline conditions; second, they must be gently mixed, otherwise the genomic DNA will break.
Solution III
The role of solution III is to precipitate proteins and neutralize the reaction. Potassium acetate is formed by replacing potassium ions in SDS with potassium ions, because sodium dodecylsulfate becomes potassium dodecylsulfate (PSS) when it encounters potassium ions. PDS is insoluble in water, and an SDS molecule combines two amino acids on average. The large amount of precipitate produced by potassium and sodium ion exchange naturally precipitates most of the protein. 2 M acetic acid is used to neutralize NaOH. Once the genomic DNA is broken, as long as it is a 50-100 kb fragment, there is no way to be co-precipitated by PDS, so the time of alkali treatment should be short, and it should not be violently shaken. Otherwise, there will always be a large amount of the resulting plasmid. Genomic DNA was mixed in and a thick total DNA band was observed on agarose gel electrophoresis. 75% alcohol is mainly used to clean the salt and inhibit Dnase; at the same time, the strong acidity of the solution III is also to make the DNA better bind to the silicate membrane.
2. Plasmid extraction step
(1) Refer to the operating instructions of the specific kit when extracting the plasmid using the plasmid extraction kit. Such as Omega's EZNA® Plasmid Mini Kit I, Q (capless) Spin (plasmid extraction cassette).
(2) Alkaline lysis hand-held method: This method is suitable for the extraction of small amount of plasmid DNA, and the extracted plasmid DNA can be directly used for enzyme digestion, PCR amplification, and silver staining sequence analysis. Methods as below:
1% of plasmid-containing E. coli cells were seeded in 2 ml of LB medium.
Incubate at 237 ° C overnight.
3 Take 1.5 ml of the cells in an Ep tube (centrifuge tube), centrifuge at 4000 rpm for 3 min, and discard the supernatant.
4 Add 0.lml of solution I (1% glucose, 50 mM / LEDTA pH 8.0, 25 mM / LTris-HCl pH 8.0) and mix well.
5 Add 0.2ml solution II (0.2mM / L NaOH, 1% SDS), gently invert and mix, placed in an ice bath for 5min.
6 Add 0.15m1 pre-cooling solution III (5mol / LKAc, pH 4.8), gently mix and mix, placed in an ice bath for 5min.
7 Centrifuge at 10,000 rpm for 20 min and take the supernatant to another new Ep tube.
8 Add an equal volume of isoamyl alcohol, mix and let stand for 10 min.
9 Centrifuge at 10,000 rpm for 20 min and discard the supernatant.
10 Wash once with 0.5 ml of 70% ethanol and drain all the liquid.
11 After the precipitate is dried, it is dissolved in 50 ul of TE buffer (or deionized water is incubated at 60 ° C).
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