Experimental principle
Nowadays, there are three commonly used plasmid extraction methods: alkaline lysis, boiling and detergent lysis. The first two methods are more intense, suitable for smaller plasmids (<15Kb), while the detergent lysis method is more gentle. It is generally used for plasmids with a larger molecular weight (>15Kb).
Alkaline lysis is one of the most widely used methods for preparing plasmid DNA. The principle is that the chromosomal DNA is much larger than the plasmid DNA, and the chromosomal DNA is a linear molecule, and the plasmid is a covalently closed circular molecule. When the pH is 12.0-12.6, the linear chromosomal DNA of the macromolecule is completely denatured and cannot be renatured in an alkaline environment, and the covalently closed-loop plasmid DNA can restore its natural conformation after adjusting the pH to neutral; In the presence of salt concentration, chromosomal DNA and protein form a precipitate under the action of detergent SDS, while plasmid DNA is soluble, and most of the cell debris, chromosomal DNA, RNA and protein can be removed by centrifugation. In the supernatant. The residual impurities can be removed by phenol chloroform.
experiment procedure
1, experimental reagents
(1) solution I 50mMl glucose / 10mMl EDTA / 25mMl Tris-HCl, pH = 8.0;
(2) Solution II 0.2Ml NaOH / 1% SDS;
(3) Solution III 3Ml potassium acetate / 2Ml acetic acid;
(4) Isopropyl alcohol, anhydrous ethanol, 75% ethanol should be pre-cooled in a refrigerator at -20 °C;
Phenol chloroform is pre-stored in a refrigerator at 4 °C;
(5) Shake fungus, 2-3 ml of the corresponding resistant LB medium, cultured at 37 ° C overnight.
2, plasmid extraction
(1) Centrifuge the solution at 12000 rcf for 5 min, discard the supernatant, add 0.2 ml of solution I (already added RNase), mix well, add 0.25 ml of solution II, mix by inversion, place at room temperature for 5 min, add 0.4 ml of solution III, After mixing upside down, centrifuge at 13,000 rcf for 30 min.
(2) Transfer the supernatant to a new 1.5 ml EP tube, add an equal volume of phenol chloroform mixture, and mix well. Centrifuge at 10000 rcf for 10 min.
(3) Take the supernatant, transfer to a new EP tube, add an equal volume of pre-chilled isopropanol, mix well, and centrifuge at 13000 rcf for 6 min.
(4) The supernatant was decanted, and 1 ml of pre-cooled 75% ethanol was used, inverted, and centrifuged at 13,000 rcf for 1 min.
(5) The supernatant was decanted, and 1 ml of pre-cooled 75% ethanol was added along the wall and directly poured off.
(6) Add 1 ml of pre-cooled ethanol along the wall and pour it directly. After inverting for 5 min, it was left at room temperature for 10 min.
After the liquid in the tube is cleaned, 100 ul of water is added to the tube and incubated at 55 ° C for 5 min.
(7) The obtained plasmid solution can be stored at -20 ° C for a long period of time.
Precautions
- If the strain is a Gram-positive bacterium, the bacterium has a layer of cell walls, and lysozyme must be added to dissolve it.
- The speed of the shaker should not be too high. It can reach OD600 1.5.
- After solution I is added to RNase, it needs to be stored at 4 ° C to prevent enzyme inactivation.
- Before using the solution II and the solution III, it is necessary to observe whether there is a precipitate. If there is a precipitate, it can be treated in a water bath at 37 °C.
- The time of solution II treatment of the bacteria should be controlled within 5 minutes.
Common problems and solutions
common problem | the reason | Solution |
Low plasmid concentration | Insufficient solution treatment | Increase the amount of solution I/II/III added, or reduce the volume of the bacteria solution. |
Low plasmid copy number | Increase the amount of shaken bacteria and reduce the final dissolved volume | |
Bacterial aging | Re-plasmid transformation or scribing to pick a monoclonal shake | |
Ethanol residue | The presence of ethanol affects the dissolution and recovery of the plasmid, and the evaporation time of the ethanol must be extended. | |
Unsuitable eluent | Configurable EB (10mM Tris-HCl, 1mM EDTA, pH 8.5) | |
Small elution volume | Increase the volume of the eluent while maintaining the concentration of the plasmid | |
Plasmid has a miscellaneous band | Bacterial contamination | Re-plasmid transformation or scribing to pick a monoclonal shake |
Reagent contamination | The plasmid extraction solution is reconfigured, and finally the solution is autoclaved and used. |
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