Canary 1

Kristina Canary
Life Science Academy
Mrs. Mountjoy
Plasmid Manuscript
March 9, 2016
How to Edvotek 301

ABSTRACT
To understand the construction and cloning of recombinant DNA, Edvotek
301 was provided. In this lab, procedures were followed, and steps were
taken to ensure that the experiment and classes objectives were met. These
objectives were to help in understanding the various techniques used to
assemble and analyze DNA molecules in vitro. The basic understanding of
cloning, plasmid extraction, and restriction enzyme analysis were also
included in the lengthy lab. With lab partners picked, students followed the
same kit and watched as different results were laid out before them, aiding
them in their identification of what types of orientations cells may take on
given certain DNA.
INTRODUCTION

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Molecular biology surrounds the field of science concerning molecularlevel biological activity. A key area of molecular biology concerns
understanding how various cellular systems interact in terms of the way
DNA, RNA and protein synthesis function (Mandal, Ananya). The various
molecular mechanisms behind processes such as replication, transcription,
translation, and cell function are studied as well. The concerns of molecular
biology is in understanding how genes are transcribed into RNA and how it
translates into protein.
A key step in synthesizing recombinant DNA molecules and therefore in
cloning is covalently joining two DNA fragments that have matching ends by
the enzyme DNA Ligase (Ligation). This is called ligation, which involves
bonding one end of a 5-phosphate to the end of a 3-OH on an adjacent
strand. Ligation is usually efficient with molecules that have identical
cohesive ends. In this lab, a strand of DNA is split and then in ligation, it is
fused again once a new strand has been introduced.
In cloning, there needs to be two ligations. One joins the two linear DNA
molecules, the other circulates the recombinant DNA molecule that was
produced from the joining. DNA cloning is the starting point for many
genetic engineering approaches to biotechnology research (DNA). To clone
DNA in cells, the specific gene or piece of DNA must be isolated, or cut,
from its source with restriction enzymes and then pasted in ligation into a
DNA vector that can replicate itself. Transformation of a cell is when the DNA

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has been successfully manipulated by the processes of restriction enzymes,
ligation, and cloning.
In the Edvotek 301 standard cloning and transformation protocol, we
assembled and analyzed DNA molecules using several recombinant DNA
techniques. This lab was to help in understanding how gene cloning, plasmid
extraction, and restriction enzyme analysis works as well as find the various
ways they result.
METHODS
The Edvotek 301 standard cloning and transformation protocol listed the
steps necessary to be taken to have a successful lab. There were five
modules total with long stopping periods provided in between so that the
steps were done efficiently without unnecessary pauses during important
procedures. The basic steps of the protocol will be outlined below as well as
the various modules they were under.
Module 1 was divided into three parts, A, B and C, due to the short
amount of time available for every module of the lab. Module 1-A began with
the stock ligation reaction mixture, which had 20 ml transferred to a control
tube, while 40 ml was transferred to a T4 DNA ligase tube. Both of these
tubes were incubated for 5 minutes. Next, the T4 tube was mixed and the
samples were incubated in a 16 degree water bath for 30 minutes. Once
finished, 20 ml of the T4 tube was transferred to a tube labeled R1 for
reaction. 5 ml of gel loading solution was added to the reaction and control

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tubes and will be used for gel analysis, while T4s remaining 20 ml goes to
module 2.
In Module 1-B, the agarose gel is created following the protocol of Edvotek
301, and then the gels chambers are loaded with lane 1 being the standard
DNA marker, lane 2 as the control, and lane three as the reaction. The
amount of time the gels run will be at 125 volts for 35 to 45 minutes. Module
1-C deals with the electrophoresis gel and chambers as well, however, this
portion of the module lists steps of how to properly remove the gel from the
electrophoresis chamber and properly stain the gel.
In Module 2, DNA from the t4 tube will be added to water within a diluted
ligation reaction tube and mixed. Then, two tubes, C2 and R2 will be created
with 500 ml of ice cold calcium chloride transferred to C2. Then, 5 colonies
from the source plate are added to C2. 250 ml of this mixture is added to the
R2 tube, and 10 ml of the diluted ligation mixture is added. C2 will also get
10 ml of the control. The tubes are placed on ice, mixed, and then incubated
on ice for another 10 minutes. Next, they receive a water bath treatment for
90 seconds so that the heat shock allows for the DNA to enter the E.coli.
Once the 90 seconds are up, they are then placed on ice for 2 minutes,
recovery broth is then added, and finally theyre incubated for ten minutes in
another water bath. This last incubation period allowed for the cells to repair
their cell walls and express antibiotic resistance.

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The cells are centrifuged for 5 minutes, the supernatant from the C2 tube
is removed, and the rest is transferred to the entire surface of the control
plate. These steps are repeated for the R2 tube and ligation plate. Then after
a 10 minute waiting period, the plates are stacked, taped, and incubated
overnight.
In Module 3, 1 colony is transferred from the ligation plate to the LB/Kan
tube containing 10 ml of the liquid medium, and is then incubated overnight.
Then in module 4, pellet cells are centrifuged and put into a test tube of 200
ml of restriction digest cocktail, here the plasmid will be extracted by
Alkaline Lysis Precipitate DNA. Next, in Module 5, the cocktail will be set up
for their various digests of EcoRI, Pvul, and the double digest (Pvull/Clal).
They will be digested, and then incubated for an hour. After all these steps
are completed, they can be analyzed by Agarose Gel Electrophoresis.
RESULTS
The final product of what our group had versus what another group had.
As seen below, our gel did not yield any bands other than a faint bar from
the DNA standard marker and the supercoiled plasmid vector standard.

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The annotated images below help to identify the base pair measurements
from each gel.
Gel 1 (our group)

Appears larger
than 10,000 bp;
was not cut with
the various digests

Around
10,000
bps

Gel 2 (other group)

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Close to 6900
bps
6790
6825

6700
bps
6850
bps
6910
bps
7100

6900

Also
close to

7450
bps

Gel 1
Lane
1

Sample
DNA Standard

Result
-----------

Molecular Weight
Around 10,000

Marker
Supercoiled

Faint purple

bps
More than

Plasmid Vector

band; very close

10,000 bps; too

Standard

to chamber

close to chamber
to tell

Gel 2
Lane
1

Sample
DNA Standard

Result
-----------

Molecular Weight
6700, 6850,

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Marker

6910, 7100,
7250, 7350,

Single thin band

7450
6825

Standard
Restriction

Another single

6900

Digest Control

thin band;

Supercoiled
Plasmid Vector

shorter than
supercoiled
plasmid vector
4

standard
Faint thin line;

EcoRI Digest

Close to 6900

located close to
5
6

Pvull Digest
Pvull/Clal Double

lane 3
Dark single band
Also very faint,

Digest

single band

6790
Close to 6900

DISCUSSION
As each step presented itself, it appeared that they were done correctly
with plenty of time to perform each efficiently. However, our results showed
that there may have been some steps that were not completed properly.
Upon having to remove the supernatant in Module 2, the pellet necessary for
the following procedure may have been either nonexistent or removed via

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pipette. This may have been what compromised the results and what
produced only the two lanes on our gel. Also, the gel was much darker in
comparison to the other gels made by other groups, so the bands may have
just been harder to see and thus making it impossible to read.
These results in both our group and the others tells that ligation is a very
delicate process and sometimes a cell will either go through transformation
or not depending on a variety of factors. Not only was the results a possibility
of improper step-taking, but the DNA may not have digested because it
didnt absorb the markers as it should have. This helped to identify the likely
causes of what makes a cell cut and ligate and what doesnt.
Some real-world applications of this could be incorporating resistant genes
into a cells DNA in order for it to become resistant to specific bacteria that
may enter the body. If these steps were done properly every time, the field of
health would be able to focus and expand on the use of certain techniques to
help in bacteria and disease resistance.
LITERATURE CITED
Construction and Cloning of a Recombinant DNA. Edvotek. The
Biotechnology Education Company,
2015. Web. 9 Mar. 2016.
DNA Cloning. Biotechnology Learning Hub. The University of Waikato, 28
Nov. 2007. Web. 9 Mar.
2016.

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Ligation (Molecular Biology). What- When- How: In Depth Tutorials and
Information. What- WhenHow, n.d. Web. 9 Mar. 2016.
Mandal, Ananya, MD. What is Molecular Biology? News- Medical. AZO
Network, 16 Oct. 2014. Web. 9
Mar. 2016.