PLASMID DNA ISOLATION

AIM: To isolate plasmid DNA from transformed E. Coli

PRINCIPLE: Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids can range from 1 -1000 kbp.

  

Based on function plasmids can be of five types:

·         F/Fertility plasmid for conjugation.

·         R/Resistant plasmid which contains genes that provides resistance to antibiotics. It also helps bacteria in producing pilus.

·         Col plasmid which contain genes that code for bacteriocin (toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains)

·         Degradative plasmid which help in the digestion of unusual substances like toluene.

·         Virulence plasmid which is responsible for pathogenicity.

Bacteria contain one or more plasmids in them. Bacteria have several mechanisms to maintain high copy number of plasmids Different plasmids have different copy numbers in the cell.

·         Relaxed plasmids are maintained in multiple copies per cell.

·         Stringent plasmids have low copy number.

Plasmid DNA may appear in one of the five conformations, which run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility from slowest to fastest:

·      Nicked Open-Circular DNA has one strand cut.

·      Relaxed Circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed. 

·      Linear DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo.

·      Super coiled (or Covalently Closed-Circular) DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.

·      Super coiled Denatured DNA is like super coiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

  

Alkaline lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or other cell components such as proteins.

Bacteria containing the plasmid of interest is first grown, and then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent breaks the membrane's phospholipid bilayer and the alkali denatures proteins involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.

Sodium dodecyl sulfate (SDS or NaDS) an anionic surfactant. The molecule has a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent.

 Proteins are contaminating agents in any type of DNA isolation so as in plasmid DNA isolation also. They can interfere with the final product and result in low yield. SDS is used to denature the proteins and facilitate the DNA purification process.

Agarose gel electrophoresis is a powerful separation method frequently used to analyze plasmid DNA.

The agarose gel consists of microscopic pores that act as a molecular sieve. Samples of DNA can be loaded into wells made in the gel during molding. When an electric field is applied, the DNA molecules are separated by the pores in the gel according to their size and shape. Since DNA has a strong negative charge at neutral pH, it will migrate towards the positive electrode in the electrophoresis apparatus. The rate at which a given DNA molecule migrates through the gel depends not only on its size and shape, but also on the type of electrophoresis buffer, the gel concentration and the applied voltage. Under the conditions that will be used for this experiment, the different forms of the same plasmid DNA molecule have the following rates of migration (in decreasing order): Super coiled > linear > Nicked Circles >dimer >trimer> etc.

REQUIREMENTS:

·         Micro centrifuge.

·         Water bath (37°C).

·         Automatic micropipettes with tips.

·         95-100% isopropanol Ice.

Buffers and Solutions

·         Alkaline lysis solution I.

·         Alkaline lysis solution II.

·         Alkaline lysis solution III.

·         Antibiotic for plasmid selection.

·         Ethanol.

·         Phenol: chloroform (1:1, v/v).

·         STE.

·         TE (pH 8.0) containing 20 μg/ml RNAse A.

Media:

·         Rich medium.

PROCEDURE :

1.      Fill a micro centrifuge tube with saturated bacterial culture growth in LB broth + antibiotic. Spin tube in micro centrifuge for one min. , & make sure tubes are balanced in micro centrifuge dump supernatant and drain tube briefly on paper towel.

2.      Repeat step one in the same tube filling the tube again with more bacterial culture the purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in micro centrifuge for one min. pour off supernatant on paper towel.

3.      Add 0.2ml of ice cold solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipette.

·         Solution 1 contains glucose, tris and EDTA.

·         Glucose is added to increase the osmotic pressure outside the cells.

·         Tris is a buffering agent used to maintain a constant pH=8

·         EDTA protects the DNA from degrading enzymes (called DNAse); EDTA binds divalent cations that are necessary for DNA activity.

4.      Add 0.4ml solution 2, eppendorf tubes and inverts 5 times gently. Let tubes sit at room temperature for five minutes.

·         Solution 2 contains NaOH and SDS (detergent). The alkaline mixtures rupture the cell, and the detergent breaks apart the lipid membrane and solubilizes the cellular proteins. NaOH also denatures the DNA into single strands.

5.      Add 0.3ml ice cold solution 3, eppendorf tubes and invert 5 times gently. Incubate tubes on ice for 10 minutes.

·         Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E coli chromosomal DNA, partially renature tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in the solution

6.      Centrifuge tubes for 5 minutes. Transfer supernatant to fresh micro-centrifuge tubes using clean disposable transfer pipette. Try to avoid taking any white precipitate during the transfer. It is ok to leave a little supernatant behind to avoid accidentally taking the precipitate.

·         This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.

7.      Fill remainder of the centrifuge tubes with isopropanol. Let tubes sit at room temperature for 2 minutes.

·         Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.

8.      Centrifuge tubes for 2 minutes. A milky pellet should be at the bottom of the tube. Pour of supernatant without dumping out the pellet. Drain tubes on paper towel.

·         This fractionation step further purifies the plasmid DNA from the contaminants.

9.      Add 1ml of ice cold 70% ethanol to eppendorf tubes and mix by inverting several times. Spin tubes for 1 minute. Pour of supernatant (be careful not to dump out the pellets) and drain tubes on paper towel.

·         Ethanol helps to remove the remaining salts and SDS from the preparation.

10.   Allow tubes to dry for approximately 5 minutes. Add 50µl TE to the tube. If needed centrifuge briefly to pool TE at the bottom of the tube. DNA is ready for use and can be stored indefinitely in the freezer.

OBSERVATION: The plasmid DNA was isolated successfully and was run on an agarose gel. After this the sample was viewed under UV.

RESULT: Plasmid DNA bands were observed under the UV.

Troubleshooting:

Smeared DNA bands were observed. It must have been due to the degradation of DNA by nucleases. Nucleases should be removed.