The plasmid is used as a vector in genetic engineering.

Plasmids are circular and

double stranded extra chromosomal DNA which can replicate separately from the
larger bacterial chromosome. Plasmids are used to carry foreign genes and introduce
them into the host cell.
In this study, plasmid DNA isolated from a bacterial culture of PKEM 103 and PUC 19
by treatment with alkali and sodium dodecyl sulfate (SDS). A powerful separation
method used for plasmid DNA analysis is Agarose gel electrophoresis. the DNA
molecules can be visualized under UV light after separation by staining with a
suitable dye.
DISCUSSION
The plasmid is also known as a set of DNA chromosome. It is used widely in science
study mostly in multiple DNA combinations for gene cloning taken from bacteria and
expanding the number of existence from the DNA itself. Besides that, it is also
used to create a large amount of protein. The size of the plasmid is determined by
the types of bacteria species. The fact is the size usually comes in a smaller
shape compared to the bacterium DNA.
The most common method used to isolate plasmid DNA is the alkaline lysis. It is the
best method to be used because of its reliability, fast and also the cleanliest way
to obtain the DNA. The method can be practiced to separate the plasmid from
bacteria. However, it consists sodium dodecyl sulfate and strong base plus sodium
hydroxide.
E. coli overnight culture had to be prepared in LB for the plasmid isolation.
Centrifuging the amount of 1.0 ml of the overnight E. coli and discard its
supernatant. During centrifuging, the felcon tube cover must be facing inside
because the pellet, during rotation, it will cause the lid to be stuck. The top
part of the tube is called as supernatant and the lower part is named as a pellet.
The layer of the supernatant is aqueous and the pellet is organic. When the cell is
released from the bacterial cell, supernatant will be removed through the
centrifuge process that inhibits enzyme during the final DNA. After that, the
pellet will dry out.
Alkaline lysis solution 1 contains Tris-Cl and EDTA where it chelates magnesium and
metals. Throwing away the two cations will spoil the balancing of the membrane.
Furthermore, the two Tris-C1 and EDTA prevents from cleaving the DNA plasmid.
Alkaline lysis that contains glucose maintains its integrity and osmolality of its
cells and avoids disruption of cells. Besides that, glucose can assist in leveling
the pH value during the lysis takes place.
An amount of 400 l Alkaline lysis solution is mixed into bacterial suspension and
incubate on ice in a duration of 5 minutes.
The SDS in the alkaline lysis solution 2 is clean where itll create holes on the
cell membrane by cleaving the proteins. This also can assist in separating proteins
from the DNA. Next, it will denature the DNA cellular by forming it into linear and
separating its strands. The circular plasmid DNA becomes single stranded.
300 l of Alkaline lysis solution 3 was added and mixed by converting the tubes
several times and incubate on ice for 5 minutes. It will neutralize the alkaline in
the suspension. The results where SDS, lipids, and protein will create precipitate
due to the high concentration of salt. Adding on the potassium will remove SDS from
the bacteria. Neutralization process takes place as the DNA will be renatured and
maintain in the solution itself due to the size which is small. Since DNA consist
large molecule size, still it is unable to renature perfectly.
Transfer the supernatant into a new tube for the new process. It will be taken
because the plasmid containing. Phenol chloroform added depending on the
supernatant volume obtained. The aqueous and organic phase is mixed by vortex and
centrifuging the emulsion at desired speed for 2 minutes. Next, the supernatant
will be placed into a new tube, adding ice and centrifuging, whereas the negative
part of the DNA will be precipitate and detected on the pellet. It can be viewed
and the DNA will be washed with ethanol to remove any salt leftover and impurities.
The pellet will be dried off. This will ensure to clean any remaining RNA.
Plasmid DNA will be tested by applying agarose electrophoresis. The DNA ladder is
ready to be applied as an indicator to clarify the size of the product. Each
consists its own DNA molecule. Measurement takes place and in the result, the
higher molecular weight DNA cannot be shifted. It is due to slow migrating speed,
which is less efficient to smaller sized molecules.
The DNA movement will be affected by the DNA molecule conformation. In normal
plasmid DNA preparation, multiple shapes of DNA may be present and it will display
negatively supercoiled form as the main band and the relaxed circular form appears
as a minor band. The rate can be manipulated using various type of electrophoresis
conditions and the larger circular DNA will grow stronger. There was no band being
observed in gel electrophoresis. This is due to plasmid was kanamycin resistance.
It will fight back against the kanamycin being added into the media. It can reduce
the bacteria activity. Unprepared measurement technique might be the cause for no
band information in the agarose gel electrophoresis as the plasmid might be a
failure due to the improper technique of handling. DNA degradation might also take
place in such situation.