碱裂解法提取质粒DNA z-bo 2020-10-14 285

　　Steps to extract plasmid DNA by alkaline lysis

　　(1) Select a colony grown in LB solid medium, inoculate it in 20 ml LB (including Amp100ug/ml) liquid medium, and shake overnight at 37°C and 250mp (approximately). 12-14 hours).

　　(2) Take 1.5 ml culture medium, pour it into a 1.5 ml Eppendorff tube, and centrifuge at 12000pm for 1-2 minutes. Discard the supernatant and place the centrifuge tube upside down on toilet paper to drain as much liquid as possible.

　　(3) Resuspend the bacterial pellet in 100μl solution I (must be shaken vigorously to evenly disperse and mix the bacteria), and place it at room temperature for 5-10 minutes.

　　(4) Add 200μl of the newly prepared solution II, close the test tube mouth, invert the Eppendorf test tube several times quickly and gently to mix the contents (do not shake), and bathe in ice for 5 minutes to achieve the effect of cell membrane. (Solution II is a dissolving solution), so the bacterial liquid in the centrifuge tube gradually becomes clear).

　　(5) Add 150μl of pre-cooled solution III, tightly cover the mouth of the tube, gently invert the tube several times to mix, and check for white cotton-like sediment. You can put it on ice for 5 minutes. Centrifuge at 12000pm for 10 minutes. Solution III is a neutralizing solution. At this time, the plasmid DNA is regenerated, the chromosome and protein form an irreversible insoluble complex, and K + precipitates the SDS-protein complex.

　　(6) Transfer the supernatant to a clean Eppendorff tube, add the same amount of phenol/chloroform/isoamyl alcohol, shake well and centrifuge at 12000pm for 10 minutes. (450μl phenol/chloroform/iso 96 penta)

　　(7) Carefully transfer the supernatant to a new microcentrifuge tube, add twice the amount of pre-cooled absolute ethanol, mix well and place at room temperature. Centrifuge for 2-5 minutes at 12000pm x 10 minutes.

　　(8) Discard the supernatant, open the test tube and place it on toilet paper to drain all the liquid, add 1 ml of 70% ethanol to wash the precipitate and centrifuge at 12000pm for 5 minutes. ..

　　(9) Aspirate the supernatant, put the test tube upside down and place it on toilet paper, drain the liquid, and let it dry at room temperature.

　　(10) Dissolve the precipitate in 20μl TE buffer (pH 8.0, containing 20μg/mlnaseA, about 4μl), and soak in water at 37°C for 30 minutes to degrade RNA molecules at -20°C. Store in the refrigerator.

　　2.10.2 Experimental reagents and preparations:

　　(1) LB liquid medium

　　Weigh 10 g trypsin, 5 g yeast extract (yeast extract), 10 gaCl, dissolve in 800 ml distilled water, and adjust the pH value with NaOH. Adjust 7.5, add water until the total volume is 1 liter, and steam sterilize under high pressure for 15 minutes.

　　(2) Ampicillin (Ampicillin, Amp) mother liquor: make a 100 mg/ml aqueous solution and store it at -20°C for later use. (3) Solution I: 50mM glucose, 25mM Tris-HCl (pH8.0), 10mM EDTA (pH8.0). Preparation method: Add ddH 2 O to 12.5ml 1MTris-HCl (pH8.0), 10ml 0.5MEDTA (pH8.0), 4.730g glucose and 500ml. Autoclave at 121°C for 15 minutes, then store at 4°C.

　　(4) Solution II 0.2M NaOH, 1% SDS

　　Preparation method: Add 1ml of 2MNaOH, 1ml of 10% SDS, and 10ml of ddH2O. Temporary setting before use. (5) Solution III: potassium potassium acetate (KAc) buffer, pH 4.8

　　Preparation method: Add ddH2O to 300 ml 5MKAc, 57.5 ml glacial acetic acid and 500 ml. Store at 4°C for later use.

　　(6) phenol/chlorine foam/isoamyl alcohol (25:24:1)

　　Chlorine foam helps to denature protein and separate the liquid phase from the organic phase. Isoamyl alcohol can eliminate the foam extraction process. Both phenol and chloroform are highly corrosive, and gloves are required during operation.

　　(7) Anhydrous ethanol

　　(8) 70% ethanol

　　(9) TE: 10mM Tris-HCl (pH8.0), 1mM MEDTA (pH8.0). Preparation method: add ddH2O to 1ml 1M Tris-HCl (pH8.0), 0.2ml 0.5MEDTA (pH8.0), 100ml. Autoclave at 121°C for 20 minutes and store at 4°C for later use.

　　1MTris-HCl (Tris (tris (trimethylol) aminomethane) preparation: Dissolve 121 g Tris base in 800 ml H2O, adjust the pH with concentrated hydrochloric acid, mix well and add water to 1L.

　　0.5 MEDTA (ethylene diamine tetraacetic acid): Dissolve 186.1g Na2EDTA.2H2O in 700ml H2O, adjust the pH to 8.0 with 10M NaOH (about 50ml is required), and then add H2O to 1L.

　　(10) RNase A mother liquor: dissolve RNase A in 10 mmol/LTris? Make a 10 mg/ml solution in Cl (pH 7.5), 15 mmol/LaCl, and heat at 100°C for 15 minutes. After cooling, use 1.5 ml Eppendorff tube to break it into small pieces and store at -20°C.