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Label all samples before putting them into wells. Fill a micropipette with sample A (it must be filled from end to end). Insert a micropipette into the glass holder. Immerse the end of pipette below the surface of the TBE and gently dispense its contents into the well (first well from the left).
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Cell transformation is the genetic difference of a cell caused from the uptake of DNA. It is most common in bacteria (bacterial transformation) and occurs naturally in some species. It can also be affected by artificial means (for example: different temperature, different chemicals such as CaCl2 i.e. Calcium chloride transformation). Cell transformation is also used to insert a new genetic material into non-bacterial cells including animal and plant cells.
Vector is a DNA molecule which is used as a vehicle to transfer foreign genetic material into another cell. There are various types of vectors such as plasmids, bacteriophages, cosmids (have large amount to store DNA as compare to plasmids) and artificial chromosomes.
The choice of vector is important because it affects so many of the processes such as cloning which includes expression, protein processing. Types of vectors and how they work varies. For example, plasmid vectors are used to multiply or express particular genes. They act as a vehicle to transfer genetic material into host cells. Viral vectors are designed for permanent incorporation of the inserting material into the human genome. These vectors leave genetic markers in the hose genome after incorporating the gene. Comparing viral and plasmid vectors, viral vectors cannot be used to multiply genes. That is because; host in a plasmid vector is immuned to have a reaction to virus.
Viral vectors can be used for gene therapy; providing a way to cure genetic disorders such as cystic fibrosis. Because these diseases result from mutations in the DNA sequence for specific genes, gene therapy trials have used viruses to deliver unmutated copies of these genes to the cells of the patientââ‚¬â„¢s body ââ‚¬” this has been very successful. However, several problems of viral gene therapy must be overcome before it gains widespread use. Immune response to viruses not only inhibits the delivery of genes to target cells but can cause health risks for the patient. Plasmid vectors can also be used for gene therapy because some methods of gene therapy depends on the efficient insertion of genes at the appropriate chromosomal target sites within the human genome, without causing cell injury or mutations (cancer).
Bacterial cell transformation is a process by which the genetic content of bacterial cell is changed. In this process, DNA is introduced into bacterial cells. Bacteria which have ability to take up foreign DNA are known as competent cells and they are made competent through use of calcium chloride. That is because; the membrane of bacterial cell is permeable to chloride ions. When chloride ions enter the bacterial cell, water molecules get attached with charged particles. This causes the cells to swell. The CaCl2 treatment (to make cells competent) is followed by heat or heat shock (at 42oC); a new set of genes (also known as heat shock genes) is expressed. This set of genes help the bacteria in surviving at such or low temperatures. Heat shock is necessary for the uptake of DNA because at temperatures above 42oC, bacteria start to lose ability to uptake DNA.
Bacteria cell transformation
How is the recombinant plasmid created? And how was it put into the bacteria?
Explain the diagram you include (Remember: How + Why (for M2)
Polymerase Chain Reaction
PCR (Polymerase Chain Reaction) is a technique used for the amplification of a small quantity of DNA over one million fold. This technique was first used to diagnose sickle cell anaemia and is now used for cloning and paternity testing. To perform DNA amplification, PCR machines are used. PCR machine helps to prepare DNA and in a short time, it increases the amount of DNA to billions.
PCR reaction has been done using a thermal cycler (the vast majority of PCR methods use cycling). PCR reaction involves different stages such as control reaction (initial stage), cycling and so on.
3 Tubes (0.5 ml)
Tube (for PCR reaction)
DNA template for amplification
10x gel loading solution
Enzyme grade ultrapure water
InstStain Methylene Blue
Distilled/Deionized water (optional)
Reason of Usage
Primers are (short strands of mRNA binded by complementary base pairs) are bonded to each DNA strand. Primer mix is a powerful tool which helps to copy every DNA sequences. It contains primers which decrease the chances to target the wrong sites on DNA. They are required to start the process of making DNA.
DNA template means pattern of DNA (to be amplified). When DNA is taken apart between the nitrogen bases, then each side acts as a pattern for the parts (such as complementary strands) that are missing. DNA template is used for amplification of DNA.
These are the genetic building blocks which make DNA molecules. These are used to create billion copies of DNA.
Initialising: DNA sample is heated at 940C -960C for 1-9 minutes.
To break the hydrogen bonds in the couple-stranded DNA, creating single-stranded molecules that are susceptible to copying. This is called denaturing. The longer the strand to be copied, the longer the denaturing process lasts.
At this stage, the temperature is lowered to
40oC-65oC for about 20-45 seconds. This allows annealing of the primers to the single-stranded DNA template. The primers are short DNA strands, designed to bond to sites at the beginning and end of the segment to be copied. If the primers are incorrectly designed or the temperature at this stage is wrong, the primer will bind randomly to the DNA, resulting in the wrong segment copy.
At this stage, 72oC-80oC temperature (optimum temperature) is used because of DNA polymerase i.e. Taq Polymerase (it is an enzyme which is used to make a new copy of DNA). This activates DNA polymerase. When DNA polymerase finds a primer (attached to a single DNA strand), it adds nucleotides on to the strand. It continues to do this until it reaches to the end of the strand and falls off.
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There is a possibility of DNA contamination in preparing a PCR sample. For example, using a same pipette to add different components or using the same tip for different components. But precautions can be taken to reduce the risk of DNA contamination such as using new pipette and tip for each different component. Wearing gloves and safety goggles can help prevent DNA contamination. Washing used equipment or discarding equipment such as used tips.
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