ISOLATION OF PLASMID DNA
Many bacterial cells contain self-replicating, extra chromosomal DNA molecules called plasmids. This form of DNA is closed, circular, double-stranded, and much smaller than chromosomal DNA; its molecular weight ranges from 2*10^6 to 20*10^6 which corresponds to between 3000 and 30,000 base pairs. Plasmids are widely used as cloning vehicles in the preparation of recombinant DNA. Bacterial plasmids normally contain genetic information for the translation of proteins that confer a specialized and some- times protective characteristic (phenotype) on the organism. Examples of these characteristics include enzyme systems that degrade antibiotics, and enzymes necessary for the production of antibodies and toxins. Plasmids are replicated in the cell by one of two possible modes. Stringent replicated plasmids are present in only a few copies and relaxed replicated plasmids are present in many copies, sometimes up to 200. Some relaxed plasmids continue to be produced even after the antibiotic chloramphenicol is used to inhibit chromosomal DNA synthesis in the host cell. Under those conditions, many copies of the plasmid DNA may be produced (up to 2000 or 3000), and may compose up to 30 to 40% of the total cellular DNA. The extensive use of plasmid DNA as a cloning vehicle often requires the isolation and characterization of plasmids. Characterization of plasmids might be necessary for any of the following reasons.
1. Construction of new recombinant DNA.
2. Analysis of molecular size by agarose gel electrophoresis.
3. Electrophoretic analysis of restriction enzyme digests and construction of a restriction enzyme map.
4. Sequence analysis of nucleotides by the Sanger or Maxam-Gilbert method.
Several methods for isolating plasmid DNA have been developed; some lead to a more highly purified product than others. All isolation methods have the same objective—separation of plasmid DNA from chromosomal DNA.
Plasmid DNA has two major structural differences from chromosomal DNA.
1. Plasmid DNA is almost always extracted in a covalently closed circular form, whereas isolated chromosomal DNA usually consists of sheared linear fragments, and,
2. Plasmid DNA is much smaller than chromosomal DNA. The structural differences cause physicochemical differences that can be exploited to separate the two types of DNA molecules.
Methods for isolating plasmid DNA fall into three major categories:
1. Methods that rely on specific interaction between plasmid DNA and a solid support. Examples are adsorption to nitrocellulose micro filters and hydroxyapatite columns.
2. Methods that cause selective precipitation of chromosomal DNA by various agents. These methods exploit the relative resistance of covalently closed circular DNA to extremes of pH, temperature, or other denaturing agents.
3. Methods based on differences in sedimentation behavior between the two types of DNA. This is the approach of choice if highly purified plasmid DNA is required. Two widely used isolation procedures based on Method 2 are described here. Both procedures yield plasmid DNA that is sufficiently pure for size analysis by agarose gel electrophoresis and for digestion by restriction enzymes.
Separation of Plasmid DNA by Boiling (Holmes and Quigley) the total cellular DNA must first be released by lysis of the bacterial cells. This is brought about by incubation with the enzyme lysozyme in the presence of reagents that inhibit nucleases. Chromosomal DNA is then separated from the plasmid DNA by boiling the lysis mixture for a brief period, followed by centrifugation. In contrast to closed circular plasmid DNA, linear chromosomal DNA becomes irreversibly denatured by heating and forms an insoluble gel, which sediments during centrifugation. Even though plasmid DNA may become partially denatured during boiling, the closed circular helix reforms upon cooling. The boiling serves a second purpose, that of denaturing deoxyribonucleases and other proteins.
Micro scale Isolation of Plasmids by Alkaline Lysis Often it is necessary to detect and analyze plasmid DNA in a large number of small bacterial samples. One widely used micro scale method is the alkaline lysis procedure. For this procedure, host bacterial cells harboring the plasmids are grown in small culture volumes (1–5 mL) or in single colonies on agar plates. The cells are lysed and their contents denatured by alkaline sodium dodecyl sulfate (SDS). Proteins and high-molecular-weight chromosomal DNA denatured under these conditions precipitate as a gel that can be centrifuged from the supernatant, which contains plasmid DNA and bacterial RNA.
1. Take 1.5ml of the culture. Culture should be prepared one day before so that the development of bacterium would be better. Check the OD of the LB broth as blank. Then take the culture solution to check the OD to know the extent of the bacterial growth.
2. Use the centrifuge, spin the vials containing culture solution spin it for 5 minutes at 6000rpm at 4᾽c.
3. After centrifugation, discard the supernatant.
4. Resuspend the pellets in 200μl of solution 1 & 400μl of solution 2.
- Glucose – 50 mM
- Tris –HCl buffer – 25mM (pH 8)
- EDTA – 10Mm (pH 8)
- 0.2N NaOH
- 1% SDS
NOTE: Because of use of NaOH, this method of ALKALINE LYSIS METHOD.
5. Mix gently and centrifuge for 5 minutes at 6000rpm.
6. Add 200μl of solution 3. Mix gently & incubate at 4᾽c for 5 minutes.
- 3M sodium acetate pH 5.2.
8. Add 600μl of iso propanol and mix gently by inverting the vials.
9. Discard the supernatant and add 70% of 100μl of ethanol. DOESN’T mix the solution to dissolve the pellet, rather, directly keep the vials for centrifugation.
10. Centrifuge at 10000rpm for 10 minutes at room temperature.
11. Discard the supernatant completely and dry the vial so that traces of the alcohol should not be there.
12. Then dissolve the pellet by using 100μl TE buffer ( pellet is very less may be just a dot like).
13. dissolve the pellets completely and load 20μl the solution on to a electrophoretic gel and proceed with electrophoresis.
14. Use the UV illuminator to check the extent sample which is moved on the gel.
check for the band pattern of plasmid.
VERY IMPORTANT NOTE:
Add Ethidium bromide directly to hot gel ( before it gets solidify) so that this compound can give colour when exposed to UV rays.
1. Use appropriate micro pipettes to transfer the solutions.
2. Use different tips each time for transferring and adding a particular solution.
3. Vials, tips, and distilled water used to prepare the chemicals must be autoclaved.
EPPENDORF TUBE (VIALS)
TIPS OF DIFFERENT SIZE
LOADING DYE IS ADDED AT THE FINAL STEP ALONG WITH TE BUFFER
GEL PATTERN AFTER LOADING
SET UP OF ELECTROPHORETIC SYSTEM
BANDS OBSERVED WITH UV ILLUMINATOR 1
BANDS OBSERVED WITH UV ILLUMINATOR 2
Band pattern shows multiple bands due to the presence of genomic DNA & trace amount of RNA.