Principles and steps of plasmid extraction, plasmid extraction method

The extraction principle is genetic recombination.

Look at this, we all use this method.

From the specific operation method, it can be divided into alkali cracking method, boiling method, toothpick method, etc.

Under the action of sodium hydroxide (alkaline environment) and detergent SDS, bacterial proteins are denatured, cells are broken, bacterial chromosome DNA DNA is denatured, double-stranded DNA hydrogen bonds are broken, and the DNA double helix structure is destroyed and deformed.

However, due to the small relative molecular weight of plasmid DNA, the DNA of the common valent closed-loop is still in a topologically winding state although denatured, and has a circular supercoiled structure, and the two complementary strands will not be completely separated even under high alkaline pH conditions.

When the pH is adjusted to neutral and there is high salt, the variant plasmid DNA returns to its original configuration, while most of the chromosomal DNA and proteins are difficult to reduce, forming insoluble complexes with cell debris, proteins, SDS, etc.

Through centrifugation and precipitation, cells are fragmented, chromosomal DNA and most proteins can be removed, while plasmid DNA and RNA with relatively small molecular mass are still soluble. The mixed RNA can be eliminated by RNA enzyme and then treated with phenol chloroform to remove residual protein. In the presence of salt and ethanol, further centrifugation precipitates plasmid DNA.

Procedure. (Please bring your own 50ml centrifuge tube, 20ml centrifuge tube, isopropanol, 70% ethanol).

1.Take 50ml of bacterial solution, put it in a 50ml centrifuge tube, centrifuge at 4000rpm for 5 min, and completely discard the supernatant.

2.Add 20ml of solution A and shake well for 2min to fully suspend the bacteria to ensure that no flocs exist. Centrifuge at 4000rpm for 5min and completely discard the supernatant.

3.Add 2ml of solution I and 40 L of solution B and shake well for 2min to fully suspend the bacteria and ensure that no flocs exist.

4.Add 4ml of Solution II, close the lid tightly, gently invert and mix 6 times, taking care that the entire inner surface of the tube needs to be in contact with Solution II, and leave at room temperature until the solution is clear. (Generally, it is 4-5min, if the solution does not become clear after 5min, it means that there are too many bacteria, and the amount of bacterial solution should be reduced.)

Be careful to mix gently, not violently).

5.Add 3ml of solution iii, close the tube tightly, mix 8 times with gentle inversion at once, and leave 4 for 5min. (In this step, be careful to mix as soon as possible, but the technique should be gentle).

6.Centrifuge at 10,000 rpm for 4 min for 15 min, carefully aspirate the supernatant and gently transfer to a new 20 ml centrifuge tube. (The centrifuge tube should be removed after the centrifuge is automatically stopped to avoid precipitated suspension).

7.Add 6ml of isopropanol, mix well, and leave at room temperature for 10min.

8.Centrifuge at room temperature at 10,000 rpm for 15 min, carefully aspirate the supernatant, and place the centrifuge tube upside down on absorbent paper to suck off the residual liquid. (The centrifuge tube should be removed after the centrifuge automatically stops to avoid precipitation.)

Hover) 9Add 3ml of 70% ethanol, gentle** for 30sec, centrifuge at 10,000 rpm for 10 min, and gently discard the supernatant.

10.Repeat step 9 and place upside down on dry absorbent paper for 2min.

11.Leave the centrifuge tube open at room temperature for 5 minutes and allow to dry the pellet.

12.Add 100 L of solution IV, gently** for 2min, to completely dissolve the pellet.

Precautions: 1. Solution II and Solution III If crystallization or precipitation occurs, it can be heated in a 37 water bath for a few minutes to dissolve until the liquid is restored to clarity.

2. The amount of biog plasmid extracted is related to the bacterial culture concentration, plasmid copy number and other factors. If the plasmid is a low-copy plasmid or a large plasmid larger than 10 kb, the cell size should be enlarged.

The amount to be used, while increasing the amount of Solution I, Solution B, Solution II, and Solution III proportionally.

1. Using the characteristics of chloramphenicol to inhibit chromosome replication, but not to inhibit plasmid replication, adding chloramphenicol during the culture of low-copy plasmids (such as PUC19) can greatly improve the yield.

2. The removal of RNA is first done by using RNA se. Adding a high concentration of RNA SEA (100 ug mL) to solution I, or dissolving the extracted plasmid with 25 ug of RNase MLTE, can reduce RNA retention, but neither can be completely removed.

3. The removal of protein mainly depends on the formation of insoluble k-SDS-protein complexes. Although it is possible to form more of this insoluble complex by leaving the neutralized system at 4C for a period of time, resulting in less protein residue, it has been shown that this is not necessary unless it is extracted in large quantities.

First of all, plasmid extraction can be divided into plasmid extraction, plasmid extraction, and plasmid extraction. At present, most laboratories use kits for extraction, and the corresponding kits can be selected according to the different needs of the experiment.

Plasmid extraction is mainly used for small extraction of plasmid DNA from E. coli, and the obtained plasmid can be used for conventional vector construction, which is generally suitable for bacterial solutions below 5ml.

The plasmid extraction, on the basis of the small extraction, can further remove the endotoxin in the bacterial solution, and the obtained plasmid can be used for cell transfection, and the amount of bacterial solution is 10-15ml. Plasmid extraction is the same as that of medium extraction, and a large number of plasmids can be obtained by extracting 100ml-300ml of bacterial solution at one time.

The principle of small-scale extraction of paramass pararoll DNA mainly includes:1Cells and ribosomes are lysed to release plasmid DNA.

SDS and proteinase K are commonly used to destroy cell structures, lyse fine stool cells and ribosomes, and release various DNA, including plasmid DNA, host chromosome DNA, etc. 2.Deproteinization.

The addition of an organic solvent such as phenol chloroform can remove the associated proteins and leave precipitated DNA. The addition of hematoxylin and isopropanol allows for selective precipitation of DNA, at which point the plasmid DNA and host DNA are precipitated simultaneously. 4.

Selective adsorption. Use adsorbed plasmid DNA such as a silicon column to elute the host DNA. The silicon column has different affinities for plasmid DNA and host DNA, and can selectively adsorb plasmid DNA, elute host elution, and transport residual concentrated DNA.

Elute the silicon column with a buffer such as TE buffer and collect plasmid DNA. Centrifugation can be used to concentrate and obtain a small volume of plasmid DNA solution. 6.

Optional PCR amplification. If the resulting concentration of DNA is insufficient, PCR amplification can be used to obtain a higher concentration of plasmid DNA. Therefore, this method realizes the extraction of small amounts of plasmid DNA through the principles of cell lysis, deproteinization, selective precipitation and adsorption, and achieves a higher concentration through PCR, which is the principle of small amount extraction of plasmid DNA.

P1: glucose prevents the suspension of E. coli from rapidly depositing to the bottom of the tube; EDTA is a chelating agent for divalent metal ions such as Ca and Mg, and its main purpose is to chelate divalent metal ions to achieve the activity of inhibiting DNase. RNase A can be added to digest RNA.

P2: This step is alkali treatment. Among them, NaOH is mainly used to lyse cells and release DNA, because in the case of strong alkalinity, the cell membrane undergoes a change from a double-layer membrane structure to a microcapsule structure.

SDS is used in combination with NaOH to enhance the strong alkalinity of NaOH, and SDS acts as an anionic surfactant to destroy the lipid bilayer membrane.

The role of solution III is to precipitate proteins and neutralize the reaction. Among them, potassium acetate is to replace sodium ions in SDS with potassium ions and form PDS, because sodium dodecyl sulfate becomes potassium dodecyl sulfate after encountering potassium ions, and PDS is insoluble in water, and at the same time, an SDS molecule combines two amino acids on average, and a large amount of precipitation produced by the replacement of potassium sodium ions naturally precipitates most of the proteins.

2M of acetic acid is to neutralize NaOH. Once the genomic DNA is broken, as long as it is a fragment of 50-100 kb in size, there is no way to be co-precipitated by PDS, so the alkali treatment time should be short, and it should not be violently shaken, otherwise there will always be a large number of basal foci and osmotic group DNA mixed on the final plasmid, and a dense total DNA band can be observed by agarose electrophoresis.

75% alcohol is mainly used to clean salt and inhibit DNase; At the same time, the strong acidity of Solution III is also to allow DNA to bind better to the silicate fiber membrane.

1.Pick individual colonies on an agar Petri dish and transfer to 3-10 ml LB culture medium at 37 150 rpm overnight.

2.Pipette the culture into an Eppendorf tube and centrifuge at 12,000 rpm for 1 minute, discarding the supernatant volume.

3.Suspend the bacterial pellet in 100 L solution (GTE solution) and mix well with strong shaking.

4.Add 200 L of freshly prepared solution (NaOH SDS solution), close the lid tightly and gently invert the centrifuge tube several times to mix the contents and leave on ice for 5-10 min.

5.Add 150 l of the solution (5mol l sodium acetate, gently invert several times to mix the solution well and leave on ice for 3-5 min.

4 Centrifuge for 10 min and transfer the supernatant to another centrifuge tube.

7.Add an equal volume of phenol:chloroform, mix well with shaking, 10,000 rpm, 4 centrifugation for 2 min, and transfer the supernatant to another centrifuge tube.

8.Add twice the volume of absolute ethanol to precipitate double-stranded DNA, mix well, and leave in the room for 2 min.

4 Centrifuge for 10 min.

10.Discard the supernatant, add 1ml of 70 ethanol to wash and precipitate double-stranded DNA, 10000rmp, centrifuge for 5 minutes at 4, pour all the ethanol, and aspirate the residual ethanol at the nozzle.

11.Add 40 g ml of RNase TE buffer to dissolve the DNA.