Introduction of Purification of Plasmid DNA by Column Centrifugation
Experimental principle
1. Spin column structure: special silicon matrix adsorption material, specifically adsorbing DNA, while RNA and protein pass through.
Under normal conditions, the surface of the nucleic acid is covered with a hydrophilic film composed of water molecules to maintain its water solubility. The addition of high concentration of salt ions destroys the relatively ordered arrangement of the hydrophilic film on the surface of the nucleic acid, forming a hydrophobic environment. In this environment, nucleic acids and silica gel membranes can bind efficiently, while proteins, metabolites and other contaminants cannot bind.
2. The principle of alkali denaturing extraction is that under the alkaline condition of pH higher than 12.6, the hydrogen bond cleavage of chromosomal DNA destroys the base pairing, resulting in the unfolding and denaturation of the double helix structure, but the closed-loop plasmid DNA strand is at Topological winding states cannot be separated from each other. When the pH is adjusted to neutral, the plasmid DNA strand quickly returns to its original configuration and remains in solution. The denatured chromosomal DNA can no longer be refolded, and it can be separated by centrifugation together with unstable macromolecular RNA and protein-SDS complex.
Experimental reagent
1. The kit comes with solution I, II, III
2. Rinse
3. Binding buffer
4. Elution buffer
5. TE buffer
Laboratory equipment
Adsorption column
2. Take the liquid gun
3. Centrifuge
4. Low pressure electrophoresis system
5. Horizontal electrophoresis tank
6. UV Transmitter
Experimental Materials
Escherichia coli with plasmid
Experimental procedure
1. Culture the bacteria: inoculate the plasmid-containing Escherichia coli into 1.5-3 ml of liquid medium containing the corresponding antibiotic, and incubate at 37 ° C for 12 to 16 hours;
2. Take 3 ml of liquid culture solution (1.5 ml X2) in an Eppendorf tube, centrifuge at 10,000 r/min for 1 min, remove the supernatant, add 100 ml of solution I, mix well; place on ice;
3. Add 150ml of solution II, cover it by inverting 6-7 times, mix it, and place it on ice for 1-2min;
4. Add 150 ml of solution III, cover and invert 6-7 times, mix and place on ice for 5 min;
5. Centrifuge at 12000r/min for 10min using a benchtop high speed centrifuge;
6. Add 420 ul of binding buffer to the adsorption column, transfer the supernatant from step 5 to the adsorption column, cover the lid of the collection tube, mix and centrifuge at 12000 r/min for 1 min;
7. Remove the adsorption column, pour off the liquid in the collection tube, put the adsorption column back into the same collection tube, add 600ul of the rinse solution to the adsorption column, let stand for 1 min, centrifuge at 12000r/min for 30 s;
8. Repeat step 7 once;
9. Remove the adsorption column, pour off the liquid in the collection tube, put the adsorption column back into the same collection tube, and centrifuge at 12000r/min for 2min;
10. Place the column into a new 1.5 ML centrifuge tube and add 40 ul of elution buffer or TE buffer to the center of the column for 3-5 minutes at room temperature. The lid was capped at 12,000 rpm for 1 minute, and a plasmid DNA solution was collected.
11. Take 5 ul electrophoresis (5 ul dye added to 100 ml gel).
Precautions
The air in the tip of the gun should be drained during loading to avoid agitation.
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