Analysis of DNA Genome Separation via Agarose Gel Electrophoresis Lab

This lab report details an experiment on DNA genome separation using agarose gel electrophoresis. The experiment aimed to understand how DNA genomes of varying sizes are separated based on their molecular weight and charge when subjected to an electric field within an agarose gel matrix. The results revealed three forms of DNA expression: digested pUC19, undigested pUC19, and circular forms. The study highlights the effectiveness of agarose gel electrophoresis in separating nucleic acids and visualizing DNA fragments using ethidium bromide staining. Factors such as gel concentration, voltage, and DNA conformation influence the separation rate. The discussion covers the importance of restriction enzymes in digesting DNA, the transformation efficiency of pUC19 plasmids, and the presence of different plasmid forms (linear, open circular, and supercoiled). The experiment confirms that electrophoresis is a reliable method for separating and analyzing DNA genomes, with implications for understanding genetic variability and enzyme activity. Desklib provides access to similar documents and study resources for students.

Analysis of Linear DNA Genomes separation in Agarose Gel Electrophoresis
STUDENT NAME
LAB REPORT

Abstract
DNA electrophoresis processes are high mobile and have high dependency on the
matrix level it takes place. The DNA gel separation undertaken in this experimental
study, shows that electrophoresis are effective in separating agarose gell and other
compound gels which contain various aspects such as polymers. DNA genomes are
determined by interplay of sieving media of the respective molecule, electric field
size and the interactions on the DNA during electrophoresis process. This
experimental design has revealed that agarose gel transformation is an important
process of separating various strand of DNA. The results of this experiment show
that there are three forms of expression of genome DNA that is the digested pUC19,
undigested PUC19 and the various lanes which exhibit circular forms. These results
signify the importance of gel DNA electrophoresis in understanding DNA genomes.

Introduction
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.

Materials and methods
Refer to the Lab Manual 5 for in-depth methodology and procedure.
Results
i) Diagrammatic presentation of gel DNA
Table 1; Showing gel electrophoresis picture
ii) Standard curve for DNA ladder

Table 2; Showing curve presentation of the base pairs against distance travelled
iii) 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
iv) Calculation process
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
v) 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 100,200,300, 700,800,900 2000,2500, 5000,6000

pUC19 400, 500,600 ,
1000,1500
3000,4000, 8000,10000
Size of insert 0.1kbp 0.2kbp 0.3kbp 0.4kbp
Table 5; Showing the sizes of pUC19 and their insert sizes
Discussion
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.
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.