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Ethidium bromide is an intercalator, meaning it inserts itself between the base pairs of DNA. Linear DNA pieces like the genomic DNA fragments bind more ethidium bromide than the circular plasmid DNA. The solution is placed into a tube that is spun extremely fast (roughly 50,000 revolutions per minute) in an ultracentrifuge for about a day. During this time the cesium chloride forms a gradient of lower density at the top of the tube and higher density at the bottom. The genomic and plasmid DNA form tight bands in this gradient. Since the plasmid DNA binds less ethidium bromide it is more dense and is located lower in the tube than the genomic DNA. RNA forms a separate band at the bottom of the tube. These three bands can be visualized by UV light.
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Role of peg in plasmid DNA isolation?
PEG (polyethylene glycol) is commonly used in plasmid DNA isolation to precipitate the DNA. When mixed with DNA in a high-salt buffer, PEG causes the DNA to aggregate and precipitate out of solution. This allows for separation of the plasmid DNA from other cellular components, making it easier to purify the DNA.
Role of ethidiumbromide in gel electrophoresis?
Ethidium bromide is commonly used in gel electrophoresis to visualize DNA under UV light. It intercalates between DNA base pairs, causing the DNA to fluoresce when exposed to UV light. This allows researchers to see and analyze DNA bands in the gel.
What is the role of bromophenol blue is plasmid DNA isolation?
Bromophenol blue is the tracking dye in electrophoresis. Being of small molecular size, it races towards the other electrode before the DNA. It is used so that you don't mistakenly let the DNA get washed off the gel and into the buffer solution.
Identify the component of the bacterial cell that is a ring-shaped piece of DNA that is NOT part of the main chromosome and often plays a role in antibiotic resistance?
The component of the bacterial cell that fits this description is a plasmid. Plasmids are small, circular DNA molecules that replicate independently of the main bacterial chromosome. They can carry genes for antibiotic resistance and other advantageous traits that can be passed between bacteria.
What is orf in plasmid vector and role in cloning of gene of interest?
ORF stands for Open Reading Frame, which is a sequence of nucleotides that can be translated into a protein. In a plasmid vector, an ORF can be used to clone a gene of interest by inserting the gene sequence into the ORF region, allowing the production of the corresponding protein. The ORF acts as a template for protein synthesis, enabling the expression of the cloned gene in a host organism.
What is the role of Cscl and ethidium bromide in plasmid purification?
The CsCl forms a gradient and the molecules migrate according to their density until they float at their individual isopycnic points (the point in the gradient that equals the buoyant density of the molecule). However, plasmid DNA and contaminating chromosome have about the same density and cannot be separated easily. This is rectified, however, by the addition of ethidium bromide. Density is a function of AT/GC ratio, but it is also a function of conformation. For supercoiled DNA, there is more DNA per unit volume than for relaxed DNA. Intercalation of ethidium bromide into DNA causes the helix to unwind (negative supercoiling) and become more relaxed. However, negative supercoiling only relaxes the DNA to a point. Further unwinding induces supercoiling in the opposite direction. When the DNA is circular and the ends are connected, the plasmid "kinks up" into a very tight knot. Thus, ethidium bromide causes the plasmid density to be increased.
Role of Glacial acetic acid in plasmid isolation?
Glacial acetic acid is used in plasmid isolation to precipitate proteins during the process of plasmid DNA purification. It helps separate the plasmid DNA from proteins, RNA, and other contaminants, allowing for the collection of purified plasmid DNA. Additionally, acetic acid helps maintain the pH of the solution, facilitating the precipitation of contaminants while keeping the plasmid DNA soluble.
Role of chloroform in plasmid isolation?
Chloroform is commonly used in plasmid isolation to separate different components in a cell lysate, such as proteins, RNA, and DNA. It helps to denature proteins and disrupt cell membranes, allowing for the separation of plasmid DNA from other cellular components. Chloroform also aids in the removal of lipids and other contaminants during the purification process.
What is the role of EtBr in electrophoresis?
Ethidium bromide interchalates with DNA. It doesn't affect electrophoresis, but it help visualise the DNA bands after electrophoresis. The EtBr that is bound to the DNA will fluoresce under ultraviolet light.
What is the Role of RNAase in plasmid preparation?
RNAase is used in plasmid preparation to degrade RNA contaminants present in the sample. This helps to ensure that the isolated plasmid DNA is free from RNA, which can interfere with downstream applications such as PCR or cloning. RNAase treatment is an important step to obtain high-quality plasmid DNA.
Role of Sodium acetate in plasmid isolation?
For DNA to precipitate down when ethanol added it needs a higher salt concentration which will allow it to precipitate more accurately, hence this salt is given in form of Na acetate which is the best salt for the purpose or else NaCl
Role of peg in plasmid DNA isolation?
PEG (polyethylene glycol) is commonly used in plasmid DNA isolation to precipitate the DNA. When mixed with DNA in a high-salt buffer, PEG causes the DNA to aggregate and precipitate out of solution. This allows for separation of the plasmid DNA from other cellular components, making it easier to purify the DNA.
Role of NaOH in plasmid DNA isolation?
Plasmid isolation involves growing the plasmid under conditions that are suitable for genes to come into play. For example the gene for ampicillin resistance; the bacteria with plasmids are placed with ampicillin so their genes can be seen for those who survived. Sodium hydroxide acts a detergent in the extraction process. A detergent's main role is to break down cell walls and cell membranes. How so? They act as poking holes into membranes. However, for the isolation of plasmid, the NaOH acts
What is the role of ECORI?
It splices the genome or plasmid in a specific location (EcoRI).
Why do you use Tris Hcl in plasmid isolation?
The role that tris-HCI plays in plasmid isolation is to maintain the pH of the solution. This prevents degradation of the plasmids. Tris stands for the organic compound, tris(hydroxymethyl)aminomethane, which is a common pH buffer. HCl is a salt acid called hydrochloride. This is added as a buffer as well to add stabilization.
What is a self-transmissible plasmid?
A self-transmissible plasmid is a type of plasmid that can transfer genetic material from one bacterium to another through a process called conjugation. This plasmid carries the necessary genes for forming a conjugative pilus and transferring the plasmid DNA. Self-transmissible plasmids play a significant role in horizontal gene transfer among bacteria.
What is the role of EcoRI enzyme?
It splices the genome or plasmid in a specific location (EcoRI).
