Gel electrophoresis is a method for separation and analysis of

macromolecules (DNA, RNA and proteins) and their fragments

based on their size and charge. It is used to separate a mix
population of DNA and RNA by length, to estimate the size of
DNA and RNA fragments or to separate the proteins by charge.
From the picture above, lane number 1 is known as the DNA
ladder, which forms bands, is used to determine the size and
quantity of testing DNA fragments by comparing to the bands
at DNA ladder. Then, for lane number 2, it acts as a negative
control which shows the negative result of the testing DNA
fragments by comparing to it.
https://en.wikipedia.org/wiki/Gel_electrophoresis

http://www.exptec.com/DNA%20ladders%20and%20DNA
%20markers.htm(Expression Technologies Inc.)
According to the result from the experiment, the DNA
fragment at lane number 3 is supercoiled. Whereas, the DNA
fragment from lane number 4 to 7 are linearized plasmids
which have the size of 4361 bases .Lastly, the 2 DNA fragments
of lane number 8 are linearized plasmids which the longer one
has the size of 3609 bases and the shorter on containing 752
bases. There are 3 lanes that are differing from the expected
result which are lane number 3(single digestion), 6(double
digestion) and 7(double digestion).
First of all, the result obtained for lane number 3 is supercoiled
plasmid which means that the plasmid is uncut (circular). This
may due to the technical error during the experiment for
example without pipetting the enzyme (EcoR1) to the DNA
plasmid. The expected result is that the DNA plasmid is in
linearized form and has the size of 4361 after the single
digestion of EcoR1 enzyme. At lane number 6 and 7, one
linearized plasmid is obtained rather than two linearized
plasmid which are expected to be the outcome. This error occur
maybe because of the absent of the second enzyme (either
EcoR1 or Pst1) or the enzyme do not cut the DNA plasmid
properly.

Besides, the results obtained for lane number 4, 5 and 8 are

similar to the expected result. The DNA plasmids at lane
number 4 and 5, which undergo single digestion, are linearized
plasmids with the size of 4361 each. This is due to the enzyme
Pst1 which cut the pBR322 at position 3607.Other than that,
the two linearized DNA plasmids produced are the product of
double digestion by Pst1 and EcoR1 where the size of the
longer linearized plasmid is 3609 and 752 base is the shorter
one.
Furthermore, nucleic acid molecules are separated by applying
an electric field to move the negatively charged molecules
through a matrix of agarose or other substances. Shorter
molecules move faster and migrate farther than longer one
because shorter molecules migrate more easily through the
pores of the gel. This phenomenon is called sieving. The
agarose gel forms a matrix which allows the DNA to pass
through. When there is an electrical current placed through the
gel, with the negative electrode at the top of the gel tank, and
a positive electrode at the bottom, the DNA added will moves
through the gel matrix towards the positive electrode because
the phosphate groups, which is a part of nucleic acid, are
attracted to the positive charge. However, the larger fragments
of DNA do not find it so easy to make its way through the gel
pores, and so moves slower than the smaller fragments which
migrate more easily. This means that the DNA is then separated
according to its size; with the small fragments migrate the
quickest and closest to the positive electrode, and the larger
fragments being higher up on the gel. The shape of the DNA
plasmid will affect the movement through the agarose gel as
well where the supercoil DNA plasmid moving the fastest follow
by the normal circular DNA plasmid and the slowest is the
nicked DNA plasmid.
https://en.wikipedia.org/wiki/Gel_electrophoresis

Nevertheless, buffers in gel electrophoresis are used to

provide ions that carry a current and to maintain the pH at a
relatively constant value. There are a number of buffers used

for electrophoresis. The most common being, for nucleic acids

Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE). Many other
buffers have been proposed, e.g. lithium borate, which is
almost never used, based on PubMed citations (LB), iso electric
histidine, pK matched goods buffers, etc.; in most cases the
purported rationale is lower current (less heat) and or matched
ion mobility, which leads to longer buffer life. Borate is
problematic; Borate can polymerize, and/or interact with cis
diols such as those found in RNA. TAE has the lowest buffering
capacity but provides the best resolution for larger DNA. This
means a lower voltage and longer time, but a better product.
LB is relatively new and is ineffective in resolving fragments
larger than 5 kbp; however, with its low conductivity, a much
higher voltage could be used, which means a shorter analysis
time for routine electrophoresis. As low as one base pair size
difference could be resolved in 3% agarose gel with an
extremely low conductivity medium (1 mM Lithium borate).
There are number of factors can affect the migration of nucleic
acids such as the dimension of the gel pores (gel
concentration), size of DNA being electrophoresed, the voltage
used, the ionic strength of the buffer, and the concentration of
intercalating dye such as ethidium bromide if used during
electrophoresis. For standard agarose gel electrophoresis,
larger molecules are resolved better using a low concentration
gel while smaller molecules separate better at high
concentration gel. High concentrations gel however requires
longer run times. Thus, the viscosity and the pore size in the
support media or gels used for electrophoresis influence the
rate of migration. Increased viscosity slows the migration and
increasing pore size speeds up the migration. The movement of
the DNA may be affected by the conformation of the DNA
molecule, for example, supercoiled DNA usually moves faster
than relaxed DNA because it is tightly coiled and hence more
compact. In a normal plasmid DNA preparation, multiple forms
of DNA may be present. Gel electrophoresis of the plasmids
would normally show the negatively supercoiled form as the

main band, while nicked DNA (open circular form) and the
relaxed closed circular form appears as minor bands. The rate
at which the various forms move however can change using
different electrophoresis conditions, and the mobility of larger
circular DNA may be more strongly affected than linear DNA by
the pore size of the gel. The rate of migration of the DNA is
proportional to the voltage applied, the higher the voltage, the
faster the DNA moves. The resolution of large DNA fragments
however is lower at high voltage.
https://www.labce.com/spg206932_rate_of_migration.aspx

https://en.wikipedia.org/wiki/Gel_electrophoresis#Agarose
A restriction enzyme or restriction endonuclease is an enzyme
that cuts DNA at or near specific recognition nucleotide
sequences known as restriction sites. Restriction enzymes are
commonly classified into three types, which differ in their
structure and whether they cut their DNA substrate at their
recognition site, or if the recognition and cleavage sites are
separate from one another. To cut DNA, all restriction enzymes
make two incisions, once through each sugar-phosphate
backbone of the DNA double helix.
These enzymes are found in bacteria and archaea and provide
a defence mechanism against invading viruses. Inside a
prokaryote, the restriction enzymes selectively cut up foreign
DNA in a process called restriction; while host DNA is protected
by a modification enzyme (a methyltransferase) that modifies
the prokaryotic DNA and blocks cleavage. Together, these two
processes form the restriction modification system.Over 3000
restriction enzymes have been studied in detail and more than
600 of these are available commercially. These enzymes are
routinely used for DNA modification in laboratories, and are a
vital tool in molecular cloning.
https://en.wikipedia.org/wiki/Restriction_enzyme

Besides, a restriction enzyme works by shape-to-shape

matching. When it comes into contact with a DNA sequence

with a shape that matches a part of the enzyme, called the

recognition site, it wraps around the DNA and causes a break in
both strands of the DNA molecule. Each restriction enzyme
recognises a different and specific recognition site or DNA
sequences which are usually only short - 4-8 nucleotides.
http://biotechlearn.org.nz/themes/dna_lab/restriction_enzymes

Loading buffer is a solution added to the electrophoresis sample

to give it colour to the naked eye. Loading buffer has two main
functions. The loading buffer contains a dense compound,
which may be glycerol, sucrose, or Ficoll, that raises the density
of the sample so that the DNA sample may sink to the bottom
of the well as DNA is hydrophilic and will dissolve in water and
If our DNA samples dissolve in the water of the electrophoresis
buffer, the DNA molecules will be spread evenly throughout the
solution and will not stay localized to the given well. The
loading buffer also include coloured dyes such as xylene cyanol
and bromophenol blue used to monitor the progress of the
electrophoresis.

http://study.com/academy/lesson/ethidium-bromide-vsloading-dye-visualizing-dna.html
SYBR Safe DNA Gel Stain is a highly sensitive stain for
visualization of DNA in agarose or acrylamide gels. SYBR Safe
stain is specifically formulated to be a less hazardous
alternative to ethidium bromide that can utilize either blue light
or UV excitation. Ethidium bromide is an intercalating agent
commonly used as a fluorescent tag (nucleic acid stain) in
molecular biology laboratories for techniques such as agarose
gel electrophoresis. When exposed to ultraviolet light, it will
fluoresce with an orange colour. Ethidium bromide is dangerous
as it act as a mutagen because it intercalates double stranded
DNA (inserts itself between the strands), deforming the DNA.
This could affect DNA biological processes, like DNA replication
and transcription. Therefore, SYBR Safe DNA Gel Stain is safer
than ethidium bromide because it has been specifically
formulated and evaluated in a battery of toxicity tests with

results indicating so that it is less mutagenic and safer than

ethidium bromide. An independent laboratory showed reduced
mutagenicity of SYBR Safe stain compared to ethidium
bromide using the Ames test.
https://www.thermofisher.com/order/catalog/product/S33102
https://www.nationaldiagnostics.com/electrophoresis/article/ethidium-bromide-staining