INTRODUCTION

To study the molecular systematics of any organism, high quality DNA is required. The rapid
availability of genomic DNA is necessary for cloning genes, selecting recombinant constructs
and for taxonomy. The cell wall is the main obstacle for quick and easy lysis of
Agrobacterium cells, and therefore, it must be disrupted for efficient recovery of genomic
DNA (gDNA). Agrobacterium utilize either enzymatic degradation followed by lysis of cells
with detergent or extraction of gDNA with phenolchloroform. For quick genotyping, cells
can also be lysed by repeated freezethaw cycles in a buffer containing SDS, followed by
extraction of gDNA with chloroform (State University of New York, n.d.). During plasmid
isolation using Quantum Prep Plasmid Miniprep kit, BioRad, the steps of resuspension,
alkaline lysis, precipitation with acidic potassium acetate and subsequent centrifugation are
performed similarly to the classical method, which the difference that the reagents supplied
by the kit manufacturer are used for the procedure. The supernatant resulting from this series
of steps will contain the plasmid DNA. This supernatant is loaded on top of a mini-column
containing a silicate-based membrane. The mini-column is placed in an Eppendorf tube so
that the flow-through can be collected upon centrifugation. Under the applied high ionic
strength conditions, the column will bind DNA molecules in the size range of 100 base pairs
to 10 kilo-base pairs. The column is subsequently washed with a wash buffer and a solution
with high ethanol content. The ethanol is then removed via repeated centrifugation. The
plasmid DNA is then dissolved in TE or a similar low ionic strength solution (Ross, Rupert,
& Paul, 1999).
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme
digestion, to quickly determine the yield and purity of a DNA isolation, and to size
fractionate DNA molecules, which then could be purified from the gel if necessary. The
agarose gel is made by dissolving the solid agarose powder in the electrophoresis buffer.
Usually Tris-AceticAcid-EDTA ("TAE") buffer is used. To make the DNA visible in the gel,
ethidium bromide is added to the gel solution and the buffer. This positively charged
polycyclic aromatic compound binds to DNA by inserting itself between the basepairs
("intercalation"). The DNA bands can be seen by exposure of the gel to ultraviolet light, due
to the the large increase in fluorescence of the ethidium bromide upon binding to the DNA.
Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis
apparatus which conducts electric current and controls the pH of the solution during
electrophoresis. DNA samples for loading into the wells ("slots") of the gel are prepared by

addition of a tracking dye which also contains a component (usually glycerol or sucrose) to
increase the density of the sample to facilitate the loading (Southeastern Louisiana
University, n.d.).
OBSERVATION
Above is a black-and-white photograph of an agarose gel. DNA fragments in the samples 1 to
11 moved from their origen, the sample wells, through the gel towards the positive electrode
thats from top to bottom in the picture.
DISCUSSION
The gel matrix acts as a sieve: smaller DNA molecules migrate faster than larger ones, so
DNA molecules of different sizes separate into distinct bands during electrophoresis. To
visualise the DNA fragments the staining agent Ethidium Bromide added to the gel and the
buffer solution. With the help of ultraviolet light the gel is exposed: DNA's as fluorescent. It
was bright colour of DNA bands due to the specific binding of the EthBr to the DNA
molecules. The sampel migration was from top to bottom: the anode (+) was at the bottom
side of this gel; the kathode (-) at the top. Smaller DNA molecules migrate faster than large
ones (Austin Peay State University, n.d.). The result, black-and-white photograph of an
agarose gel containing ethidium bromide, after electrophoresis of 11 DNA samples, the gel
was on a UV lamp when photographed. More DNA in a band gives more intense staining of
that band. So, 50l (1KB) of DNA in a band gives brighter staining as in result. Where
restriction fragments originating from one microgram of identical DNA molecules are
separated. Which means that the bands contain equimolar amounts DNA. The smallest
fragment of 500 base pairs is hardly visible, while the biggest fragment of more than 10,000
base pairs shows a very bright band. The bands differ in intensity: larger fragments bind more
EthBr. This is very good visible in the size marker (first well). All fragments in this lane are
generated by digestion of one particular DNA, so fragments are present in equimolar amounts
and the brightness of the bands corresponds to their lengths. And all the 11 samples also in
equimolar amounts which shows 10000 base pairs (1KB) single band without any extra or
double bands. It is because the method done in isolation of plasmid and preparation of
electrophoresis was correct according the standard procedure.
REFERENCES

Ross, A., Rupert, M.S., & Paul J.B. (1999). Wizard Plus Minipreps for the Isolation of Binary
Plasmids from Agrobacterium tumefaciens [PDF document]. Retrieved from:
https://www.promega.com/~/media/files/resources/promega%20notes/70/
State University of New York. (n.d.). Isolation and Electrophoresis of Plasmid DNA [PDF
document].
Retrieved
from:
http://faculty.buffalostate.edu/wadswogj/courses/BIO211%20Page/lectures/lab
%20pdf's/Plasmid06.pdf.
Southeastern Louisiana University. (n.d.). Experiment 2: Plasmid DNA Isolation, Restriction
Digestion and Gel Electrophoresis [PDF document]. Retrieved from:
http://www2.southeastern.edu/Academics/Faculty/jtemple/486/experiment%202.pdf
Austin Peay State University. (n.d.). Gel Electrophoresis of Plasmid DNA [PDF document].
Retrieved
from:
https://www.apsu.edu/sites/apsu.edu/files/chemistry/SP11_1021_GEL_ELECTROPHO
RESIS_of_Plasmid_DNA.pdf