Experimental Reagent 1. Solution I
50mmol / L glucose
25mmol / L Tris.Cl (pH8.0)
10mmol / LEDTA (pH8.0)
Solution I can be prepared in batches, about 100ml per bottle, steam sterilized under high pressure of 6.895 × 104Pa for 15 minutes, and stored at 4 ℃
2. Solution â…¡
0.2mol / L NaOH (10mol / L stock solution is diluted before use)
1% SDS
3. Solution â…¢
5mol / L potassium acetate 60ml
Glacial acetic acid 11.5ml
28.5ml of water
The prepared solution is 3 mol / L for potassium and 5 mol / L for acetate.
4. STET
0.1mol / L NaCL
10mmol / L Tris.Cl (pH8.0)
1mmol / L EDTA (pH8.0)
5% Triton X-100
5. Lysozyme solution
10mg / ml, formulated with 10mmol / L Tris.Cl (pH 8.0).
Experimental Step 1. Bacterial harvest
1) Add 2ml of LB containing the corresponding antibiotics to a 15ml volume well-ventilated (not tightly closed) test tube, then insert a single colony, and incubate overnight at 30 ° C with vigorous shaking.
2) Pour 1.5ml of culture into a microcentrifuge tube, centrifuge at 12000g for 30 seconds at 4 ° C with a microcentrifuge, and store the remaining culture at 4 ° C.
3) Aspirate the culture solution and make the bacterial pellet as dry as possible. The easy way to remove the supernatant is to use a single-use tip to connect to the vacuum tube, gently suction, and use the tip to contact the liquid surface. When the liquid is sucked from the tube, keep the tip as far as possible from the bacterial sediment, and then continue to use the tip to remove the droplets attached to the tube wall by vacuuming.
2. Alkaline cracking method
1) Precipitate the bacteria, resuspend in 100 μl of solution I pre-chilled with ice, and shake vigorously. Make sure that the bacterial pellets are completely dispersed in the solution I. The bottoms of the two microcentrifuge tubes are in contact with each other and shaken to rapidly disperse the pellets.
2) Add 200μl of freshly prepared solution II. Cover the tube tightly and quickly invert the centrifuge tube 5 times to mix the contents. Make sure that the entire inner surface of the centrifuge tube is in contact with solution II. Do not shake, place the centrifuge tube on ice.
3) Add 150μl of ice-cold solution III, close the mouth tightly, shake the tube upside down and shake it for 10 seconds. Solution III is evenly dispersed in the viscous bacterial lysate. minute.
4) Centrifuge for 5 minutes at 12,000g at 4 ° C with a microcentrifuge, and transfer the supernatant to another centrifuge tube.
5) Do it or not: add an equal amount of phenol: chloroform, mix by shaking, centrifuge at 12000g for 2 minutes at 4 ℃ with a microcentrifuge, and transfer the supernatant to another conscience tube. Some workers believe that it is not necessary to use phenol: chloroform for extraction, but for some unknown reason, omitting this step often results in DNA that can tolerate the restriction enzyme reaction.
6) Precipitate double-stranded DNA at room temperature with twice the accumulated ethanol. Mix by shaking and leave at room temperature for 2 minutes.
7) Centrifuge at 12 ° C for 5 minutes at 4 ° C with a microcentrifuge.
8) Carefully aspirate the supernatant and put the centrifuge tube upside down on a paper towel to allow all the liquid to flow out. Then remove the droplets attached to the wall of the tube. The easy way to remove the supernatant is to connect the disposable pipette tip to the vacuum tube and use the pipette tip to contact the liquid surface. When the liquid is sucked out of the tube, try to keep the tip away from the nucleic acid precipitate, and then continue to use the tip to remove the droplets attached to the tube wall by vacuuming.
9) Wash the double-stranded DNA precipitate with 1 ml of 70% ethanol at 4 ° C, remove the supernatant according to the method of step H, and allow the nucleic acid precipitate to dry in the air for 10 minutes.
Note:
1. The high copy number plasmid prepared by this method (such as Xf3 or pUC), the yield is generally about: 3-5μg per ml of original bacterial culture.
2. If you want to analyze the DNA by restriction enzyme cleavage reaction, you can take 1μl of DNA solution into another microcentrifuge tube containing 8μl of water, add 1μl of 10 × restriction enzyme buffer and 1 unit of the required restriction enzyme, incubate for 1 -2 hours. Store the remaining DNA at -20 ° C.
3. This method can be applied to 100ml bacterial culture by scaling up at an appropriate scale:
3. Boiling cracking
1) Precipitate the bacteria and resuspend in 350μl STET.
2) Add 25μl of freshly prepared lysozyme solution and shake for 3 seconds to mix. If the pH in the Huaihuai is below 8.0, the lysozyme will not be effective.
3) Place the centrifuge tube in the boiling water bath for exactly 40 seconds.
4) Centrifuge at 12 000g for 10 minutes at room temperature with a microcentrifuge.
5) Use a sterile toothpick to remove bacterial debris from the microcentrifuge tube.
6) Add 40μl of 5mol / L sodium acetate (pH5.2) and 420μl of isopropanol to the supernatant, mix by shaking, and leave at room temperature for 5 minutes.
7) Centrifuge at 12 ° C for 5 minutes at 4 ° C using a microcentrifuge to recover the nucleic acid pellet.
8) Carefully aspirate the supernatant and put the centrifuge tube upside down on a paper towel to allow all the liquid to flow out. Then remove the droplets attached to the wall of the tube. The easy way to remove the supernatant is to use a single-use tip to connect to the vacuum tube, gently suction, and use the tip to contact the liquid surface. When the liquid is sucked out of the tube, keep the tip as far as possible from the nucleic acid precipitate, and then continue to use the tip to remove the droplets attached to the tube by vacuuming.
9) Add 1ml of 70% ethanol, and centrifuge at 12,000g at 4 ° C for 2 minutes.
10) Aspirate the supernatant gently again as described in step H. This step should be handled with extreme caution, because sometimes the precipitation block is not tightly attached to the wall, remove all the ethanol droplets formed on the wall of the tube, open the tube mouth, and place at room temperature Until the ethanol has evaporated, there is no visible liquid in the tube (2-5) minutes.
11) Dissolve the nucleic acid with 50 μl of DNase-free pancreatic RNase (20 μg / ml) TE (pH 8.0) to dissolve the nucleic acid, shake it slightly, and store at -20 ° C.
Note: When a small amount of granulation, especially DNA, is obtained from an E. coli strain (endA strain, such as HB101) expressing endonuclease A, it is recommended to discard the boiling method. Because the boiling step cannot completely inactivate endonuclease A, the plasmid DNA can be degraded after incubation in the presence of Mg2 (when using restriction enzymes in V). Adding a step between step I of the above scheme, namely extraction with phenol: chloroform, can avoid this problem.
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