Isolation of Genomic DNA and Double-Stranded Plasmid DNA from Bacterial Culture with Restriction Analysis of Genomic DNA Using Universal Restriction Enzymes and DNA Extraction and Purification.
Objective:
1. To use the aseptic technique in transferring bacterial colonies to bacterial growth media.
2. To learn the technique of vortexing or pipetting in order to mix well the solutions.
3. To learn technique on how to isolate the genomic and plasmid DNA of the bacterial culture.
4. To do the Restriction Analysis using Universal Restriction Enzyme
5. To learn the technique of DNA extraction and purification from Agarose Cell.
6. To learn the technique in performing the Agarose Gel Electrophoresis.
7. To learn the skill of separating the supernatant from the pellet.
Genomic DNA is the DNA found in the chromosomes. It can be either double-stranded or single-stranded. In modern molecular biology laboratories, DNA is routinely isolated from cells as the first step in analysing genes.
In order to obtain the purified DNA from cells, scientists must first release the DNA by breaking open the cell membrane and the nuclear membrane. Once the DNA is released from the nucleus, however, it can be destroyed by enzymes in the cytoplasm that degrade DNA. As a result, there are some ways to guard the DNA against attack by enzymes from the cell. Lastly, the released DNA must be separated from the proteins, lipids, and carbohydrates, amino acids, fatty acids, and other cellular molecules.
There are two features of most DNA isolations that help protect the DNA. The first is coldness which is isolation of DNA procedure is usually done on ice to slow the action of cellular enzymes that might degrade the DNA. The second is gentleness: DNA strands are extremely long and fragile. Therefore all steps should be done slowly and gently to avoid shearing the DNA strands. The same things go for the plasmid DNA.
There are some methods to extract the plasmid DNA that is either using the spin column or the conventional methods. As we know that, bacterial cell contain both the genomic DNA and plasmid DNA, scientists need to isolate the plasmid from the genomic DNA. The most suitable procedure is used to extract plasmid DNA from bacterial cell suspensions based on the alkaline lyses procedure developed by Birnboim and Doly. Both nucleic acids and proteins denature under the alkaline conditions, and once the solution is neutralized by the addition of Potassium Acetate Salts, chromosomal DNA and proteins aggregate together while the plasmids renature correctly and remains in the solutions. This procedure take advantage of the fact that plasmids are relatively small supercoiled DNA molecules and bacterial chromosomal DNA is much larger and less supercoiled. As a results, the chromosomal DNA unable to renature back correctly.
Restriction Enzyme (or restriction endonuclease) recognizes a specific base pair sequence in DNA called a restriction site and cleaves the DNA within or near that sequence. All restriction enzymes cut DNA between the 3’ carbon and the phosphate moiety of the phosphodiester bond. It can be either in the form of sticky end or blunt end.
It is encouraged to use more than 1 restriction enzyme in the restriction digestion if both enzymes are active in the same buffer and the same incubation temperature. But in this experiment, we are just using only 1 restriction enzyme that is Pst 1. Small amount restriction enzymes are needed in the digestion solution depending on size of the DNA being digested. Larger DNAs should be digested in larger total volumes (between 50 -100µl) and vice versa. Besides that, different enzymes require different buffer solutions. Enzymes will only perform effectively in appropriate pH, salt concentration and temperature. In order to choose the appropriate assay buffer, we need to refer to the vendor’s catalog for the chart of enzyme activity.
Agarose gel electrophoresis is an easy method to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole. The plasmid was found in many different supercoiled forms in the bacteria. When plasmid was isolated from a bacterial culture, all the different supercoiled forms of the plasmid was isolated, and each will migrate differently on the gel, producing about three major bands and many minor bands. When this mixture of supercoiled plasmids is cut with a restriction enzyme, the different forms linearize and unwind. As a result they all become identical and run at the same rate, and usually only one band seen on the gel.
Elution is a method of quickly purifying agarose gel DNA fragments for use in subsequent reactions such as further restriction enzyme modification, T4 ligase, Taq polymerase, Klenow or polynucleotide kinase. Although higher yields of purified DNA can be obtained from commercially available purification kits, greater ligation efficiencies per given amount of DNA have been seen with the use of spin column.
Sterile bijou bottle
Autoclaved tips
Bunsen burner
Various sizes Eppendorf tubes (650µl, 1.5ml, 1.7ml, 2.0ml)
Sterile silanized glass wool
Hypodermic needle and syringe
Clean razor blade
Tissue paper
Yellow pipette tips
Materials:
5 ml sterile Luria Broth (LB) media
Agar plate containing single colonies of Bacillus amyloliquenfaciens UMAS 1002
Overnight Bacterial cell culture containing cloned plasmid
DNA genomic samples
Pst 1 restriction endonuclease
Cut and EtBr stained agarose gel containing DNA band
1X TE Buffer10X
Pst 1 Buffer
10mM Tris-CL pH 7.4
1mM EDTA
Sterile ddH2O
10% SDS
20mg/ml Proteinase K
5M NaCl
CTAB/NaCl solution
Chloroform/Isoamyl alcohol (24: 1)
Phenol / Chloroform / Isoamyl Alcohol (25: 24: 1)
Isopropanol
70% Ethanol
Cold Absolute ethanol
Solution I:-
50mM Glucose
1.8M Formic Acid
25mM Tris-HCl, pH8
Solution II:-
0.2M NaOH
1% SDS
Solution III:-
3M KAc (Potassium Acetate)
10mM EDTA, pH 8
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