“Experiment No: Dat Plasmid isolation by alkaline lysis method Aim: To isolate the plasmid from bacterial culture by alkaline lysis method Principle: Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes. Size of plasmids range from 1-200 kb. 4. Harvest and resuspension: Bacterial cells are harvested by centrifugation (plasmid 1. Resuspension is inside the cell), and resuspended in E.coli cell Tris.HCl buffer containing EDTA. EDTA 4° inhibits DNases by chelating divalent metals SRT % (primarily Mg?" and Ca’). Glucose, added to 2.lysis _ maintain osmolarity, prevents the cells from NoOH/S0S 4 bursting, =. - tpn ON 2. Cell lysis: Cells are lysed with NaOH and ORE 4 SDS. SDS solublize the phospholipid and <= _ protein components of the cell membrane, 3. Neutralization leading to lysis and release of the contents. NaOH denatures the chromosomal and plasmid DNAs, as well as proteins. 3. Neutralization: The lysate is neutralized 4, Clearing of by the addition of acidic potassium acetate. lysates The addition of high salt concentration causes - potassium dodecyl sulphate (KDS) to Centifugation Filtration precipitate, and denatured _ proteins, chromosomal DNA, and cellular debris are | Supernatant KDS precipitate Je salt detergent | contoining containing coprecipitated in insolubl eterg ee eh complexes. Plasmid DNA, being circular and * proteins covalently closed, renatures correctly and remains in solution. = filtrate containing 4. Clearing of lysate: Precipitated debris is | [us| ON* **]<- plasmid DNA removed by either high speed centrifugation | |). ")Shanosonel or filtration, producing cleared lysate 5. Phenol chloroform extraction (optional): Phenol-chloroform extraction removes remaining contaminant proteins from the DNA sample. When phenol-chloroform is mixed with the aqueous solution containing DNA, proteins will move into the phenol phase and will be separated from the aqueous DNA. Aqueous layer containing DNA is collected after centrifugation. 6. DNA precipitation and resuspension: DNA is precipitated by adding either cold 100% ethanol or room temperature isopropanol. The DNA pellet is collected by centrifugation, washed with 70% ethanol, air dried and resuspended in TE buffer.Reagents required 1, Resuspension buffer (Solution |; 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) Glucose 901 mg EDTA disodium salt, dihydrate: 372.24 mg Tris 302 mg Total volume : 400 ml Adjust the pH to 8.0 with HCl. 2. Lysis solution (Solution I; 0.2 N NaOH, 1% SDS) NaOH 800 mg sps 19 Total volume 100 ml Freshly prepare before use. 3. Neutralization buffer (Solution Ill; 3M KOAc, pH 6.0) Potassium acetate 29.44 g Glacial acetic acid 11.5 ml Total volume £100 ml Store at 4°C 4, Phenol:Chloroform:isoamyl (25:24:1) 5. Isopropanol 6. Cold 70% ethanol 7. TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) Tris 2121 mg EDTA disodium salt, dehydrate: 37.2 mg Total volume 100 mi Adjust the pH to 8.0 with HCl. Procedure 1. Grow overnight cultures from single colonies of bacteria (E. coll) containing the plasmid of interest. Add 1.5 ml of the bacterial culture to a 2 ml microcentrifuge tube. Centrifuge at 13000 rpm for 3 min. Pour off the supernatant, being careful not to disturb the bacterial pellet. 2RoN ‘Add in 100ul of cold resuspension buffer (Solution 1). Resuspend the pellet by vortexing for 2 min or until all bacteria are fully resuspended, 6. Add 200 ul of lysis solution (solution Il) and mix by inverting the tube 5 to 10 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured. Note: Avoid vigorous stirring or vortexing at this stage as this can shear the bacterial chromosome which will contaminate the plasmid DNA. 27. Incubate solution for 5 min at room temperature. Note: Do not allow the lysis to proceed for longer than 5 min. A § min incubation allows maximum release of plasmid DNA, while minimizing the release of chromosomal DNA and reducing the exposure of the plasmids to denaturing conditions, 8. Add 150ul of cold Solution Il! and mix the solution gently but thoroughly by inverting several times (do not vortex). A white precipitate will be formed which contains the bacterial proteins, genomic DNA and debris. 9. Incubate the tube on ice for 10 min, 10. Centrifuge the tube for 5 min at 13,000 rpm Note: Pellet contains proteins, cell fragments, salt and other extra particles from solutions. Supernatant contains the plasmid DNA separated from bacterial chromosomes. 11. Collect the supernatant into a new 1.5 mi microcentrifuge tube by carefully pouring. 12. (optional) Add 450 jul Phenol:Chloroform:lsoamyl alcohol. Vortex for 5 min and leave at room temperature for 5 min, 13. (optional) Centrifuge at 13000 rpm for 10 min and collect the aqueous upper layer in a new tube 14. Add 350 ul Isopropanol, mix well and Incubate for 10 min at room temperature. 15. Centrifuge the tube for 5 min at 13,000 rom. 16. Carefully pour off the supernatant (do not pipette) 17. Add 1 ml of 70% ethanol and gently invert the tube several times to wash the DNA pellet. 18. Centrifuge at 13,000 rpm for § min and pour off the supernatant (do not pipette) 19. Centrifuge the tube with DNA pellet for 5 sec to collect residual ethanol. 20. Remove the supernatant as much as possible with 200 ul micropipette. 21. Allow the pellet to air-dry for 10-15 minutes. You want to evaporate as much of the ethanol as Possible without letting the DNA pellet completely dry 22. Resuspend the DNA in 50 yl TE buffer. 23. To dissolve the pellet, incubate at 4°C for 30 min and mix well by micropipette. 24. After DNA has dissolved, measure the concentration by diluting 10 ul of DNA into 1 ml of TE buffer (1:100 dilution) and measure the absorbance at 260 nm and 280 nm. 25. Store the DNA at 4°C. 26. Calculate the amount of DNA using absorbance at 260 nm, and estimate the purity of the DNA by Azeo/Azso ratio. Concentration of DNA (ug/ml) = Absorbance x dilution factor (100) x 50 Results: Discussion: