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Materials and methods

Discuss about the Analysis of Linear DNA Genomes Separation in Gel Electrophoresis.

Agarose gel electrophoresis has been widely used as a form of separating DNA genomes in varying sizes from 100 kp upto 25 kb. Isolation of Agarose gel is obtained from the genera Gelidium and Gracilaria.in the gelato process, the polymers of agarose often form an association of none covalent which form networks of pore sizes which determine the molecular ability of sieving properties. Use of gel electrophoresis is beneficial in separation of DNA genomes.

Electrophoresis process is key in separating the different nucleic acids using various sizes and charges depending on the contents of the solution. In this experiment, lab analysis of gel was used to put gel solutions in charged nucleic acids for separation purposes. At this point the larger DNA and RNA have a hard time in separating thus allowing time for separation of the genomes based on the sizes. The rate of separation of the DNA molecule in the experiment was determined by the rate at which the sizes of the DNA, the concentration of the gel, DNA Conformation present, voltage degree applied, ehidium bromide solution introduced, type of agarose and the buffer being utilized in electrophoresis.

After the process of separation, DNA molecules will be able to be visualized in the UV light using staining process to identify the different genomes. Thus in essence DAN electrophoresis defines the process by which the DNA migrates in the supporting medium. Most of electrophoresis is carried in agarose gels in narrow polymers of gels using pores of different sizes, this sieving provides a means by which the pores gives an opportunity for the DNA molecules to go through the pores at different sizes thus being separated using molecular weights.

Thus this laboratory report uses agarose Gels while staining with ethidium bromide  to assess the separation process of the different DNA genomes. Thus it seeks to investigate the DNA genome separation to assess the different nucleic acids by their respective sizes.

Refer to the Lab Manual 5 for in-depth methodology and procedure.

Diagrammatic presentation of gel DNA

Table 1; Showing gel electrophoresis picture

Table 2; Showing curve presentation of the base pairs against distance travelled

  • Table of standard curve values

Bp size

100

200

300

400

Log10(bp)

2

2.3

2.3

2.4

Distance cm

0.5

0.4

0.4

0.38

Table 3; Showing table figure for the curve

Table 4; Showing how to calculate base pairs

Example suppose we have a base pair having travelled 0.3 cm, then draw a line as illustrated above and take the readings on the corresponding logbp and take the anti log, which you get the base pair size.

  • This gives us anti log of 3.0, which is 1000kbp
  1. Insert values table

Bp size

100

200

300

400

Log10(bp)

2

2.3

2.3

2.4

Distance

0.5

0.4

0.4

0.38

Size of pUC19

100,200,300,

400, 500,600

700,800,900,

1000,1500

2000,2500,

3000,4000,

5000,6000

8000,10000

Size of insert

0.1kbp

0.2kbp

0.3kbp

0.4kbp

Results

Table 5; Showing the sizes of pUC19 and their insert sizes

Agarose gel electrophoresis has been utilised as a common method for separation of proteins, (Kryndushkin et al., 2003). The basic forms of nucleic acids can be separated through the aid of electrification process whereby charged molecules move to the anode side. This migration as depicted in the experiment ensures that molecules which have lower molecular weight are able to move faster, (Sambrook & Russel 2001). The process of electrophoresis is a crucial step in ensuring purification process of the desired DNA bands. In this experiment the usage of ethidium bromide is essential in visualizing the staining of the transcend DNA molecules.

In this task, the Agarose gel electrophoresis plays a key role in ensuring the characteristics of DNA are obtained without any alterations. This experiment has yielded results which have enabled determination of DNA fragments sizes through digestion by restriction enzymes. The visualization has been effected with the use of ethidium bromide which is a common agent in nucleic acid purification process. The Agarose gel concentration on this task entailed the separation of the gel using agarose gel concentration of 0.2%w/v having bands from 0.1-1 kb.

The distance travelled by DNA molecules in electrophoresis is directly proportional to the size of the DNA itself. The agarose gel is beneficial in ensuring that there are movements based on their sizes. With the various differences between the various rates of the DNA molecules in the gel solution, they are separated based on the size of the bases. The relationship built between the varied sizes of the DNA genome. The sieving of DNA is done through the size which it bears, (Southern, 1975).

The length of DNA strands often vary from 50 base pairs to upto million s base pairs which agarose gel electrophoresis can be effective in separating them , the migration and distance travelled is linked on the concentration of the agarose used to prepare the gel. Concentrations having lower concentration are able to travel faster in the distance travelled and vice versa. In this study agarose gel of 2% has been used which was effective in separating the DNA at range of 0.1-1 kb, the low percentile gels often signify gels which are weak.

Double stranded DNA moves faster as the molecules travels; its speed is inversely proportional to the logarithm of base pairs. This linked and established relationships depends on the strength of the of gel composition. The distance travelled by the digested genome signifies that there is action of restriction enzymes which shows that there restrictions which have taken place, thus distinguishing the variability linked to genetics and enzyme cost. The digested fragments were this separated using the agarose gel electrophoresis which showed continuous smear on the gel surface with the distribution of the difference fragment sizes being established. Digested pUC19 is a plasmid and able to transform itself on the transformation process where it can be able to multiply itself and express.

Calculation process

Undigested pUC19 originate from E coli and contain high number of base pairs. The transformation efficiently portrayed shows that smaller pUC19 plasmid sin E choli can be manipulated and be transformed from the ampicilin forms. This shows that the DNA is in contact form with plasmid DNA being intact and with presence of viral chromosomes which can be transformed into high efficiencies. This transformation is through the resulting effect of digestion of peri plasmids. The undigested Puc19 shows presence base pairs which have the ability to perform recombination and be incorporated into cells, (Goto, Kenta & Yukio, 2013).

The lanes which have recombination factor is able to facilitate the cloning of DNA in host cells.  This signifies recombination of various fragments of gel solution. The lanes that have been generated originated from digestion of particular DNA, which gives it equimolar amounts. Based on the lanes, there is variation on the number of non molar amounts, thus signifying that there is difference in band lengths. Others have shown to represent circular forms of the plasmids which is dependent on the age and quality of the plasmids.

The existence of three forms of DNA formation which exists include linear formation, open circular formation and supercoiled forms. Plasmid DNA have been prevalently been studied in laboratory studies. After its preparation they exists in the three forms above. With good plasmid preparation, DNA often form plasmid which exist in any one strands of the DNA, this break causes the release of the phosphordiester backbones of the DNA to be released out.

The visualising process of the agarose gel using the standard control tool is key to assess whether the bands have created a generation or not. Closer bands are well compressed than far away bands as indicated in the gel view. The standard marker used in this experiment was essential in ensuring that the standards sizes are generated using base pairs.

This result signifies that electrophoresis is an effective way of separating nucleic acids. High gel agarose gives room for handling of low percentage gel separation. Due to the size of the base pair present in this experiment, has utilised field gel electrophoresis. This is  comparable to studies done (Lee et al, 2012), which have shown that sizes of DNA can be separated effectively through plotting on the log of molecular weight and different bands of DNA against the distance moved, this portray how different forms of gel can be able to move at different speeds. Super coiled plasmid DNA have sown to move faster, while those in linear formation travel averagely while open circular travel slowly.

References

Goto, K., & Nagano, Y. (2013). Ultra-low background DNA cloning system. PloS one, 8(2), e56530.

Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD & Kushnirov VV (2003). Yeast [PSI+]  prion aggregates are formed by small Sup35 polymers fragmented by Hsp10. Journal of Biological Chemistry.278 (49): 49636.

Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments: JoVE, (62).

Sambrook J&Russel DW(2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.

Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J mol biol, 98(3), 503-517.

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