Extraction and purification of nucleic acid is the first step in most molecular biology studies and all in recombinant DNA techniques. DNA extraction is a process of isolation of Genomic DNA from RNA, protein, and lipid. Basically, a genome is the complete genetic contents in chromosomes of eukaryotes and prokaryotes or in the RNA and DNA of viruses. Genome is the total haploid set of organism’s genes (Campbell, Reece, Taylor, Simon, & Dickey, 2011). The purpose of DNA extraction is to obtain DNA in a relatively purified form which can be used for further investigations such as PCR and DNA sequencing. Many different methods are available for isolating genomic DNA. Modification and optimization of DNA extraction methods are usually done for different types of cell (Dhaliwal, 2013). Cetyltrimethyl ammonium bromide (CTAB) is a preferable method that can be used for isolating the genomic DNA. There are four important steps in DNA extraction protocol of animal tissue which are enzymatic digestion of cellular protein, DNA precipitation, DNA washing, and DNA hydration (refer to Figure 1 in Appendix 1).
Firstly, the cellular protein is digested through enzymatic process. In this step, the animal tissue is grind until it is tiny enough. This is important to break up the cell wall since DNA is located within the nucleus in eukaryotic organisms. Then, 600µl of CTAB buffer is pipetted into the eppendorf tube and mixed well with the grind tissue. CTAB buffer (hexadecyltrimethyl ammonium bromide) acts as detergent that help to lyses the cell membrane (Herzer, 2001). Moreover, 15µl of Proteinase K is also added to the eppendorf tube. Proteinase K is used to digest contaminating proteins. According to Kim (2013), there is chances that enzyme w...
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...ompletely dry, 20µl of distilled water is added into the eppendorf tube. The function of distilled water is to re-dissolve the DNA pellet. After that, the eppendorf tube together with the DNA solution is placed at 4oC in a freezer. The DNA is kept in the DNA freezer and is taken out from the freezer when the DNA is wanted to be used to further any suitable study related to the DNA.
Works Cited
Campbell, Reece, Taylor, Simon and Dickey (2011). Biology. Petaling Jaya, MYS: Pearson Custom Publishing.
Dhaliwal A. (2013). DNA extraction and purification. Mater method, 3, 191.
Herzer S. (2001). DNA purification. Retrieved from http://biologya.ifsc.usp.br/biomplcel1/outros/DNA%20purification.pdf.
Kim J. (2013). 10 questions you want to ask about Proteinkinase K. Retrieved from http://info.agscientific.com/blog/bid/158711/10-Question-you-want-to-ask-about-PROTEINASE-K
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Firstly, samples were taken out carefully. The frozen tissues of sea cucumbers were thawed in the sink with running tap water followed by multiple washes using distilled water to remove the foreign particles. The surgical blades, surgical blade-holder, and labelled sample tubes were prepared. Then, the tissue samples were cut-sliced from each samples followed by storage in the properly labelled tubes. Each sample was stored in separate tube. Different blade was used for each sample. The obtained in-tube-samples were stored in -20 oC freezer for the next step of DNA extraction.
...q DNA polymerase to each tube while disallowing the tubes to cool and without taking
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
We first started out with DNA extraction. We swabbed a toothpick on the inside of our cheek. We isolated the mitochondrial DNA from that sample of cheek cells. We used different reagents that lysed the cells and made it easier for the students to be able to have access to the nucleic acids. For example the buffer ATL breaks down the cell membrane, but has the environment similar to the cell as far as salt and pH concentration. The proteinase K breaks do...
It was loaded into the gel instead of in the well leading to no results. There should have been a series of bands similar to well three. In lanes three, 5, and 6 we see the bands labeled A,B and C. Due to the short distance traveled down can discern that these bands are in fact DNA as it is both bulky and negatively charged. In lane seven one can see the band in the beginning, which is identified, as tRNA. E and F represent the left of “junk” from the inside of the cell that made it’s way into the sample such as mRNA and proteins. This can be told by seeing that the small materials traveled very far down the gel and were not removed by DNase. What truly tells one whether or not he or she extracted DNA are the blank spots on the gel. In lane four and eight there are missing bands. This is due to the fact in these samples enzymes where added to break down the nucleic acids, DNase in the case of lane four and RNase in eight, thus causing a gap where they should appear. The data that was collected seems to indicate that the sample that was extracted was done properly and yielded DNA. It should be noted that the lanes three and four were switched when adding the material into
To transfer the DNA to the microtubes, we used a micropipette. It is a device that sucks up the perfect amount of DNA for each tube. Also, the micropipette has disposable tips, so after each use, we can get rid of the tip so we don’t contaminate our experiment. We used a fresh tip for each sample. With a new tip each time, I grabbed of each DNA sample and placed it into the correct labelled test tube.
Paabo’s team, from Leipzig, Germany, used a method of amino acid content as a way of measuring extractible DNA from the bones. The amino acid method was a...
The use of Proteinase K and Homogenization Buffer (HB) is used to cause the cell to lyse, breaking down the lipid cell membrane, digesting unwanted proteins and removing contamination that might otherwise degrade the DNA. This allows for the DNA to become free floating.
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
This causes the DNA fragments to move through the gel depending on their sizes. With this, the DNA fragments will show a sample that will determine how large they are to one another. Gel electrophoresis uses a horizontal gel-like slab. These gels are made of polysaccharide called agarose, which is dry, powdered flakes. When the agarose is heated in a buffer, it makes the gel form solid, slightly squishy gels. (Dickey, J. L. 2012) At one end of the gel, there is square shaped space that is called wells, this is where the DNA sample will be placed. Before the DNA samples are added, the gel must be added to the well of the gel box. One end of the box is hooked to a positive electrode and the other a negative electrode. When active, the DNA samples will start to migrate from the positive to the negative showing a difference with the DNA samples with some fragments that are larger and some that are smaller. (Mader. S. S. 2016)
5 test tubes were used for this lab, each label 1-5. Then we added 1ml of assign solution for each test tube. Tube 1 had DNA, 2 had RNA, 3 had the nucleic acids from the liver extract supernatant, 4 had water, and 5 had the unknown. 2ml of Dische’s reagent was then added to each test tube. All of the test tubes are then place in boiling water. After 15 minutes in the boiling water, we recorded the color of each of the different test tubes in a table.
Next the restriction enzymes are used to cut the DNA into smaller pieces. Restriction enzymes work by cutting the DNA at a specific sequence and results in many fragments of different lengths. These fragments are called restriction fragments length polymorphisms (RFLP’s). These RFLPs are then put into an agarose gel. Using gel electrophoresis, the fragments are sorted according to size. When the current of the electric field is turned on, the negative RFLPs will start to move across the gel towards the positive end. The smaller fragments move farther across the gel than the larger ones. The gel is then covered by a piece of nylon and thin paper towels, which are used to absorb moisture from the gel. The DNA fragments get gently transferred from the gel to the surface of the nylon. This process is called blotting. Radioactive probes get washed over the nylon surface. These probes will join to any DNA fragments that share the same composition. The final step to making a genetic fingerprint is to place a photographic film on top of the nylon surface. The probes leave marks on the film wherever they attached to the RFLPs. Dark bands will then show up when the film is developed, which marks the length of the RFLPs. Then scientists are able to compare the DNA from the crime scene and the DNA from a
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...
All individuals, except identical twins, have unique DNA. DNA fingerprinting is an unambiguous identification method that takes advantage of the difference in the DNA sequence. This process of DNA fingerprinting starts with the isolation of the DNA from the identified sample, such as blood, saliva, semen or other body tissues. In instances where the available sample is small for the process of fingerprinting, then the sample is augmented through a process called polymerase chain reaction (PCR). PCR is the process of DNA replication that does not use living cells and, therefore, the process is appropriate in fingerprinting old samples. The focus of this process is on the short tandem repeats (STR) that have short units of DNA that are repeated several times in a row. After the DNA is isolated and amplified it is treated with restriction enzymes. This process cuts the DNA strands at definite sequences called restriction fragment length polymorphisms (RFLPs). Since everybody’s DNA is different the resulting RFLPs will be of different sizes. These fragments are observed in gel electrophoresis; a process that separates DNA based on the size of fragments. RFLP analysis is based on the fact that since everyone’s genetic sequence or the variable number of tandem repeats is different this result in the different sizes of RFLPs. Gel electrophoresis involve the separation of the fragments of DNA as they migrate through an agarose gel when an electric current is applied to the gel. The DNA that has separated is then drawn out of the gel with a nylon membrane which is treated to break the hydrogen bonds holding the DNA strands together. The separated strands of the DNA is then cross-linked to the nylon membrane a...