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In this article we will discuss about the principle, requirements and procedure for isolation of plasmid DNA using alkaline lysis method.
Principle:
The alkaline lysis method of plasmid DNA is based on lysis of bacteria by treating it with SDS and NaOH, neutralisation of this mixture with potassium acetate which causes rapid annealing of the plasmid, removal of chromosomal DNA and bacterial proteins by centrifugation and ethanol precipitation of reannealed plasmid.
Requirements:
1. L.B. (Luria Bertani) Medium (pH 7.0):
2. Solution I (Suspension buffer- GTE solution):
3. Solution II (Lysis buffer):
(Do not autoclave SDS). Filter sterilise through 0.45 /µm filter and store at room temperature.
4. Solution III (Potassium acetate 5M- pH 4.8):
5. RNAse 10.0 mg/ml
6. Phenol: Chloroform =1:1
7. Ethanol 70%
T.E. Buffer (pH 8.0):
Autoclave and use.
8. Reagents for agarose gel electrophoresis.
9. Laminar clear air flow hood.
10. Incubator.
11. Environmental shaker.
12. Refrigerated centrifuge.
13. Vortex mixer.
14. Micropipettes.
15. Eppendorf tubes.
16. Inoculating needles.
17. Bunsen flame.
18. Test tubes.
19. E.coli cultures.
Procedure:
(a) Growth of bacterial culture:
1. Inoculate a loopful of E.coli in 3 ml L.B. broth with 100 µ.g/ml ampicillin.
2. Incubate overnight (OD= 0.8-1.0) at 37°C on an environmental shaker at 220 rpm.
(b) Harvesting and lysis of bacterial cells:
1. Distribute 1.5 ml of culture each in two eppendorf tubes and centrifuge at 10,000 rpm for 5 minutes.
2. Pour out the supernatant completely leaving the bacterial pellet as dry as possible.
3. Suspend the pellet in 100 µl of solution I [Glucose 50 mM, Tris HCl (pH 8.0) 25 mM, EDTA (pH 8.0) 10 mM] by vortexing. Add 2 µg/ml lysozyme before adding bacterial pellet. Glucose provides an isoosmotic condition to prevent physical shock; pH of the solution is raised to basic level with Tris to help denaturing DNA, EDTA stabilises the cell membrane by binding the divalent cations.
4. Leave the tubes on ice for 5 minutes.
5. Add 200 µl of solution II (freshly prepared Lysis buffer; 0.2 (N) NaOH and 1.0% SDS). Mix GENTLY by inverting the tube and leave on ice until SDS precipitates (5 minutes) and becomes cloudy and viscous due to lysis of cells. Treat the cells GENTLY; otherwise chromosomal DNA will shear and release it from the membrane which will copurify with plasmid DNA. The detergent SDS, lyses cells and NaOH increases the pH so that DNA denatures.
6. Add 150 µl of solution III (ice cold 3 M Potassium acetate and glacial acetic acid) to lysate and mix GENTLY by inverting the tubes several times and then leave them on ice for 15 minutes. A white flocculant precipitate of SDS/ lipid/ protein complex appears. Potassium acetate returns to neutral pH and helps in the precipitation of chromosomal DNA which is now trapped in the SDS/lipid/ protein complex.
7. Centrifuge at 4°C for 10 minutes at 15000 rpm. The pellet contains cell debris and chromosomal DNA.
8. Using a micropipette, collect the supernatant, which contains plasmid DNA, into a new tube.
(c) Purification of plasmid DNA.
1. Add equal volume of phenol: chloroform (1:1) mixture, mix GENTLY by inverting the tube several times, centrifuge at 10,000 rpm for 15 minutes and remove the aqueous phase.
2. Add two volumes of absolute ethanol to the pellet, mix by vortexing, keep at room temperature for 5 minutes, centrifuge at 15000 rpm for 5 minutes at 4°C.
3. Discard supernatant, collect the pellet and dry.
4. Add 200 µl of 70% ethanol to the pellet, rinse, centrifuge, decant alcohol and dry the pellet of DNA.
5. Dissolve the pellet in 30 µl of T.E. buffer (pH 8.0) with 1 of RNAse (10 mg/ ml), incubate at 37°C for 30 minutes. RNA ase destroys RNA present in the pellet.
6. Redissolve the final pellet of DNA in T. E. buffer. (10 mM. Tris 1 mM EDTA pH 8.0) and estimate concentration of DNA.
7. Check plasmid DNA preparation by loading on 1% agarose gel.