CONSTRUCTION AND CLONING OF A RECOMBINANT DNA

Emily Linn and Lucy Kurtz

ABSTRACT
In this lab, we used DNA cloning technology to replicate the kanr gene for kanamycin
resistance. This technology is a relatively new and extremely relevant process in biomedical
research today. Paul Berg did the first successful application of the technology in 1971. In our
study, we used the steps outlined in the Edvotek 301 standard cloning and transformation
protocol. We found success in each step with the exception of Module II and Module V. Our
DNA was successfully recombined, however, the digest results were inconclusive according to
the possible orientations.
INTRODUCTION
Genetic cloning technology is a relatively new and extremely relevant process in
biomedical research today. The first successful application of recombinant DNA technology was
in 1971, when Paul Berg successfully spliced bacterial lamba DNA into the DNA of simian virus
SV40 (BioTech, 2016). The process of digestion and ligation employed by Berg is the same
technology used in this cloning experiment. Plasmids, usually made up of 2,000 to 10,000 base
pairs, are digested by a restriction enzyme, and a fragment is typically inserted between the
sticky ends where the cut is made (Slish, 1999). Between insertion and replication, the samples
of digested DNA must be run through a gel to determine success. Then, DNA ligases are added
to the sample that catalyze the formation of a phosphodiester bond between the 3' hydroxyl and
5' phosphate of the adjacent DNA residues (BioLabs, 2016). The plasmid is then left to replicate
within the bacteria as it goes through binary fission. Recombinant DNA works when the host cell

expresses protein from the recombinant genes (Kuure-Kinsey et. all, 2000). For this reason,
bacteria must be tested to ensure that only those that contain the vector survive. They are placed
on plates where only antibiotic-resistant bacteria grow, and the only bacteria with resistance are
those that took up the inserted fragment as well. The cloned genes can then be collected in a lab.
In Our Study, we used E. coli vectors. These plasmids are commonly used because they can be
simplified by reducing their length (Griffiths et. all, 1999). The fragment that we are adding to be
replicated is the kanr gene for kanamycin resistance.

METHODS

We used the Edvotek 301 standard cloning and transformation protocol. The basic steps
of the protocol are outlined below:
o Module I
I-A: Ligation of the Plasmid Vector to the kan^R Gene Fragment
Stock ligation reaction mixture is added to a ligation control tube (C1) and

a T4 DNA ligase tube.

Sample are incubated and half of DNA ligase is added to ligation reaction

tube (R1).
Loading gel solution is added to C1 and R1 to be analyzed on a gel.
T4 DNA ligase tube moves to Module II for transformation.
I-B: Agarose Gel Electrophoresis
Agarose gel is made.
I-C: Staining with InstaStain Ethidium Bromide
Gel is stained with FlashBlue stain.
Gel is analyzed once stain is finished.
o Module II: Transformation of the Recombinant DNA into E.coli
E.coli colonies are added to CaCl2 and suspended into the solution.
Solution is then transferred to a ligation reaction tube (R2) and a control
reaction tube (C2).

Diluted Ligation Rxn DNA is added to R2 and control DNA is added to

C2.
R2 and C2 are spread on two plates for colonies to form.
o Module III: Culturing of kan^R Transformants
One single isolated colony is added to a liquid medium containing
Kanamycin antibiotic.
Solution is then incubated overnight and a pellet is formed.
o Module IV: Extraction of Recombinant Plasmid DNA
Buffer is added to the pellet and pellet is resuspended into the solution.
A lysis buffer is then added to the solution and then mixed.
Potassium Acetrate Solution is added to the mixture and then mixed to

form a white precipitate.

Supernatant is transferred to a clean tube and 100% isopropanol is added

and mixed.
Solution is centrifuged to form pellet and then washed.
Pellet is resuspended in a 1x Tris-EDTA buffer.
o Module V
V-A: Restriction Enzyme Analysis
Restriction Digest Cocktail is made and added to tubes 3, 4, 5, and 6.
Ultra-pure water is added to each tube.
EcoRI is added to tube 4.
PvuII is added to tube 5.
ClaI is added to tube 6.
Tubes are prepared for electrophoresis.
V-B: Analysis of Restriction Digests by Electrophoresis
DNA samples are added to the wells and electrophoresis is ran using he
same steps in Module I-C.
RESULTS
o Module I
R1 and C1 tubes to be analyzed on a gel

T4 DNA Ligase tube to be sent to transformation

A stained final gel with ligation results

o Module II

o Module III

A control plate with colonies and ligation reaction plate with colonies

o Module IV

Tube containing bacterial colonies and Kanamycin solution

Tube with resuspended pellet in a 1x Tris-EDTA buffer

o Module V
4 tubes
3 Control
4 EcoRI
5 PvuII
6 ClaI
Gel with final solution of cloned DNA

Lane

Sample

Result

DNA Standard
Marker

Molecular Weight
(bp)
6751, 3652,
2827,1568, 1118,
825, 630

Supercoiled
Plasmid Vector

4300

Standard
Restriction Digest
Control
EcoRI Digest
PvuII Digest
PvuII/ClaI

3
4
5
6

3600

2
1
2

4250, 3500
4000
3650, 3500

DISCUSSION
Each step was successful with the exception of Module II and Module V. We believe we
either did not have enough E.coli in our tubes and we incorrectly spread the solution on the
plates. Therefore, there was not any growth on our plates. Our final gel had bands incongruent
with the restriction digest most likely because of digestion at incorrect temperatures and/or
inappropriate amounts of time. However, we believe it was closest to a Single Insert 5-3
orientation. This technology is typically used in labs in a more intensive form. The gene we
inserted only codes for antibiotic resistance, and is usually used in standard lab procedures as a
tell for successful recombination of another target DNA sequence. These sequences typically
code for proteins or enzymes of medical importance, such as insulin. Final products can either be
transcribed for pharmaceutical companies or further amplified for scientific use.

LITERATURE CITED

BioLabs. 2016. DNA Ligation. https://www.neb.com/applications/cloning-and-synthetic-

biology/dna-ligation. Date accessed (03/09.16).

BioTech. 2016. Recombinant DNA. https://biotechhistory.org/timeline/recombinant-dna/.

Date accessed (03/09.16).

Griffiths, A.J.F., W.M. Gilbert, J.H. Miller, and R.C. Lewontin. 1999. Making
Recombinant DNA - Modern Genetic Analysis.

http://www.ncbi.nlm.nih.gov/books/NBK21407/. Date accessed (03/09.16).

Kuure-Kinsey, M. and B. McCooey. The Basics of Recombinant DNA.
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html. Date

accessed (03/09.16).
Slish, D. 1999. Cell Biology Lab.
http://faculty.plattsburgh.edu/donald.slish/LabSyllabus.html. Date accessed (03/09.16).