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Introduction

What is a plasmid?
A plasmid is a circular double stranded structure of DNA molecule which is separate from a cell’s own chromosomal DNA. Plasmids are found in bacteria, yeast and some eukaryotic organisms. Plasmids range from a few thousand base pairs to 100 kilobases (kb). When an organism containing plasmid divides,

the plasmid is also replicated before every cellular division. Gel Electrophoresis Plasmids exist in a symbiotic relationship with their host. They provide some benefit to the host especially resistance from antibiotics. Drug resistance plasmids make their host resistance to the mechanisms of several drugs. Plasmids also contain transfer genes which code for certain proteins and can be transferred from one organism of the same species to another. This transfer results in widespread resistance of the bacteria or other species to a drug in a certain setting such as a hospital (Wilson and Hunt, 2002).
Spectrophotometric analysis of DNA

The amount of nucleic acid in a sample can be checked by the use of UV spectrophotometry. DNA and RNA both absorb UV light effectively which makes it possible for the analysis to detect the presence of nucleic acid even if the concentration is extremely low. The spectrophotometric analysis uses the principle that nucleic acids absorb UV light in a specific pattern and if present, the emergent rays striking the photodetector will be less than the incidence rays and hence produce a higher optical density. The value of the optical density obtained will depend on the concentration of the DNA or RNA present in the

sample (Huss, Festl and Schleifer, 1983).
Gel electrophoresis, how can the plasmid DNA be visualized on an agarose gel?
Gel electrophoresis is a technique which is used to separate DNA according to its size for the purpose of visualization and purification. The procedure requires an electric field which moves the negatively charged nucleic acid towards the positively charged electrode. The fragments of DNA move towards the electrode at the speed depending on their molecular weight. Hence the shorter DNA fragments will move faster along the gel matrix than the ones longer in size.

Therefore the exact length of each fragment can be determined by running them alongside a DNA ladder which contains DNA fragments of known lengths. In order to visual the DNA, ethidium bromide is added. When it binds to the DNA fragments it allows the user to visualise the fragments under UV light (Addgene, 2015).

What is a restriction enzyme?
In order to clone a specific part of the DNA present in a plasmid molecule, the fragments must be extracted out and then inserted into vector DNA. Restriction enzymes are present in the bacteria which recognise certain sequences of nucleic acid which are known as restriction sites. The enzymes then cleave the DNA at those specific sites.

These enzymes cleave the nucleic acid from within the DNA molecule and hence are also known as restriction endonucleases (Wilson and Hunt, 2002).

What is a restriction map?
A restriction map is a map which contains known restriction sites in a sequence of a DNA. Restriction enzymes can be used to form a restriction map. They are used in the process of gel electrophoresis as a reference to extract plasmids (Dale and Schantz, 2002).
Aims of experiments
The aim of experiment is to isolate and mapping of bacterial plasmid DNA.

Method
The experiments were carried out as described in the schedule that given at Coventry university.
1. Isolate plasmid DNA by a rapid alkaline lysis method.
2. Isolate plasmid DNA using a commercial kit
3. Analyse the plasmid DNA by agarose gel electrophoresis.
4. Use spectrophotometry to analyse the purity and yield of nucleic acids
5. Restriction endonuclease digestion of DNA and the analysis of the results of such digests.

Results

Experiment 4: Isolation of the plasmid
Isolation of plasmid is a method which is used to extract and purify the plasmid present in DNA. First the bacteria known to contain the plasmid are grown in a culture medium under favourable conditions. The purpose of this step is to increase the number of bacteria so that the DNA can be extracted from them. The grown bacteria are then lysed under alkaline conditions which denature the proteins and DNA content of the sample. However the plasmid present remains stable. Neutralization buffer is added which precipitates the denatured proteins and DNA and are filtered out. The plasmid stays in the solution, hence being isolated (Bimboim and Doly, 1979).
Analysis of the gel

Distance moved (mm) from well
Measured on gel picture. Fragment size (bp)
3 5000
4 2000
6 850
8 400
9 100

Table 1 shows the fragment size and distance moved of the five fragments which were obtained from the sample and ran on agarose gel.

Graph 1 shows the fragment size and distance moved of the five fragments which were obtained from the sample and ran on agarose gel.

Experiment 5: Spectrophotometric analysis

The spectrophotometric analysis shows that there are 6 different bands.

Experiment 6: Restriction digest
It is a procedure used to prepare DNA in order to process it for analysis. It cleaves the DNA at specific site. The procedure uses specific enzymes known as restriction enzymes. The enzymes EcoR1, Sal 1, PVU11 and Pst1 were used.

Discussion
Plasmid DNA yield extracted in each preparation using the n-butanol protocol in our experience was very low. By doubling homogenization time, increases yield. Glass beads can be added before the homogenization in order to achieve rapid breakdown of the substrate and enhanced plasmid yield. Impurities in DNA can lead to inaccurate results in the measurements of DNA concentration. To improve accuracy of results and accommodate for impurities in the solution, the turbidity can be adjusted. Impurities could be genomic DNA or chemicals used in the process of extraction of DNA.

The purpose of the calibration curve is to provide a set of reference points which can be used to compare unknown chemical samples. During analysis of certain samples, it is possible to achieve a complete and accurate make-up of the structure present in the sample hence a calibration curve can help to compare it with known standards of information which allow estimation of the unknown sample at hand.

Some of the fragment sizes which were obtained did not add up to the expected size. This issue could be due to two factors, either the extremely small DNA fragments run off the gel or that the gel was not thick or dense enough in order to resolve one fragment from the other which were of similar size. The plasmid can be either in linear or supercoiled or circular state. Supercoiled DNA travels faster and further due to its type of confirmation. The linear plasmid is achieved when the restriction enzyme is able to cut the DNA at both ends of the plasmid. It travels at the rate which is predicted…