CHEMICAL AND BIOMOLECULAR ENGINEERING

LAB /
BIOENGINEERING LAB

CH1801
BG1801
Plasmid Isolation

Location: N1.2 B2-41

Name: Brandon Lam Chi Ee
Matric Number: U2220508G
Group: CBE 02 / Group 3
Date of experiment: 31 August 2022
1. Introduction
1.1. Introduction to the Escherichia Coli bacteria
Escherichia Coli, or E. Coli, is a bacterium that is commonly found in the lower intestine
of warm-blooded animals (World Health Organization, 2018). It is also commonly used
in laboratory settings as a model organism for microbiology and recombinant DNA
technology, due to the ease of E. Coli cell growth in terms of cell growth speed and
growth requirement (Science Learning Hub, 2014).
E. Coli is used widely used to produce heterologous proteins, which are proteins not
expressed naturally by the host, but rather inserted synthetically via plasmids in a
laboratory setting for the expression of the desired proteins. As a result of E. coli fast
growth rate and the ability to express proteins via plasmids, it is one of the most
preferred hosts for protein production (Cronan, 2001).
1.2. Plasmids
Plasmids are circular, extrachromosomal DNA that can be present in bacteria cells.
These DNA are not essential for the main function of the bacteria but may benefit the
host bacteria in many ways through expression of the protein the plasmid carries
through its coding region. One such example is antibiotic resistant gene, which is one
part of any plasmid that when coded, allows the cell to express a protein which allows
it to be resistant against a certain antibiotic, hence allowing the survival of the bacteria
against the antibiotics.
Recombinant plasmids can be engineered in vitro to include the desired coding region
to produce the desired protein, and hence transforming the bacteria into a recombinant
vector. Essentially, the bacteria are being “tricked” to produce the protein of interest
that the user wants. This protein is the desired product of the biopharmaceutical
industry, and hence is studied as a recombinant technology.

2. Theory
Selection
To retrieve plasmid DNA from the cell culture, E. Coli, one must be able to identify the
bacteria that carries the plasmid of interest. To ensure that the selection of cell culture
carries the plasmid of interest, plasmids that are synthesized in vitro contain a
selection marker that identifies the bacteria to carry the plasmid. As stated before, one
such selection marker is the antibiotic resistant gene. Therefore, bacteria that carries
the plasmid which in turn carries the antibiotic resistant gene, will be resistant to the
antibiotic. The antibiotic used in this experiment is ampicillin.
When ampicillin is applied to the cell culture, only the bacteria that contains the
recombinant plasmid will survive, and therefore can be easily identified and selected
for plasmid isolation.

1
 Figure 1: Plasmid vector for gene transfer with selectable marker (Jeltsch, 2016)

DNA extraction and separation

After isolating the cells that contains the plasmid vector, the plasmid vector must be
extracted and separated from cellular contents. To do this, a hypotonic sucrose buffer
is added, to increase the cell size. This is due to the hypotonic sucrose buffer causing
the intracellular pressure to be much greater than the extracellular pressure, allowing
water to enter the cell and expanding its volume, hence allowing the cell to swell and
rupture. This buffer is also known as Buffer P1 in the context of this experiment.
Afterwards, sodium dodecyl sulfate (SDS) supplemented with ribonuclease (RNase)
is added as Buffer P2. SDS is an alkaline detergent, and its primary function is to
disrupt the cell membrane by dissolving phospholipid bilayer and denaturing cellular
protein, while also denaturing the plasmid and chromosomal DNA present in the E.
Coli cell by breaking the hydrogen bonds present in the nucleotides. RNase is a protein
enzyme that helps to cleave the RNA molecules into smaller pieces, such as mRNA
and tRNA present in the cell, hence eliminating them. This reaction should not proceed
more than 5 minutes as to prevent all the plasmid DNA from being denatured.
Buffer N3, a high salt potassium acetate, is added to neutralise Buffer P2 and
essentially stop the reaction. This proceed renatures the plasmid DNA as
complementary strands rehybridises to the original hydrogen bonds, allowing it to
remain in the solution. However, chromosomal DNA is not renatured, but captured
together with a white precipitate containing the SDS from Buffer P2, denatured protein
as well as all other cellular contains such as the lipids from the phospholipid bilayer.
(G Bio Sciences, n.d.)
Via centrifugation, the solution containing plasmid DNA is removed as a supernatant
while the white precipitate is then discarded.
Plasmid DNA Filtration
Plasmid DNA is transferred to a QIAprep column, a silica-based resin column that
allows filtration of plasmid DNA via the selective adsorption of the plasmid DNA
binding to the resin, in the presence of a high chaotropic salt (QIAGEN, n.d.). The
solution is centrifuged to hasten the binding of plasmid DNA to the resin and the flow-
through is discarded.

2
Buffer PE is added to the resin to wash down the column and elute any particulates
that does not have any binding to the resin but is present in it. After centrifugation to
remove residual wash buffer, Buffer EB, an elution buffer, is added to elute the
plasmid DNA from the column, and the flow-through is collected as a solution of
plasmid DNA.
Spectrophotometric method
To determine the purity of plasmid DNA that is collected, spectrophotometry is
performed.
The underlying principle of spectrophotometry is that nucleic acid absorbs ultraviolet
(UV) light strongly at wavelength 260 um, due to their molecular structure. By
measuring intensity of UV light that is passed through the plasmid DNA solution
through a spectrophotometer, the optical density of the solution can be obtained as
A260. By principle, since nucleic acid strongly absorbs UV light of wavelength 260 nm,
the less light is captured by the spectrophotometer, the higher the concentration of
nucleic acid in the solution.
The same principle is applied for wavelength of 280 um is used to determine the purity
of protein, as protein has a strong affinity at wavelength 280 um, as A280. Determining
the ratio of wavelength A260/A280, is an indication of the purity of nucleic acid against
proteins, with ratio of 1.8 – 2.0 to be of good ratio of purified DNA.

3. Procedures
3.1. Preparation of E. Coli culture for DNA extraction
Day 1
1. 25 mL of Luria-Bertani (LB) broth is mixed with 500 μL of ampillicin.
2. Using an inoculating loop, 1 scoop of a speck of E. Coli is mixed with the LB
broth containing ampillicin.
3. The cell culture is then transferred to a bacteria incubator shaker, where it is
incubated at 37°C for 16-18 hours, shaking at 250 RPM.
Day 2
4. The incubated cell culture is transferred into 2 x 1.5 ml of Eppendorf tube.
5. The Eppendorf tube is then transferred into a micro centrifuge machine and
spun at 13000 rpm for 2 minutes.
6. The supernatant is disposed, and the bacteria pallet is ready for DNA
extraction.
3.2. Plasmid DNA Extraction (For 1 x cell culture)
Lysis of cell culture
1. The bacteria pallet is resuspended by the addition of 250 μL of chilled Buffer
P1. It is then vortexed at top speed, to ensure homogeneity of the solution.

3
2. 250 μL of Buffer P2 is added, and the tube is inverted 4-6 to gently mix the
solution. The lysis reaction is allowed to continue no longer than 5 minutes.
3. 350 μL of Buffer N3 is added and the tube is then inverted to mix the solution.
Plasmid DNA Filtration
4. The solution is then placed into a microcentrifuge to spin at 11,000 RPM
(default spin setting) for 10 minutes.
5. The spun solution is observed to have separated into two distinct area: as
shown in Figure 2 below. The supernatant is collected with a micropipette, with
emphasis to avoid the white particulate, and placed into a QIAprep column,
which is also placed into a collection tube, as shown in Figure 3.

Figure 2: White precipitation with solution Figure 3: QIAprep column

6. The column is centrifuged for 1 minute and the flow-through is discarded.

7. 750 μL of Buffer PE is added into the column and solution is centrifuged for 1
minute. Flow-through and collection tube is discarded, and QIAprep column is
placed into new collection tube.
8. 50 μL of Buffer EB is added to elute plasmid DNA from the column, and solution
is allowed to settle for 1 more minute.
9. The column is centrifuged once more for 1 minute. QIAprep column is discarded
and flow-through is obtained as a solution of plasmid DNA.

3.3. Plasmid DNA purity measurement

Quartz cuvette preparation
1. Two plasmid DNA solution are mixed in one collection tube, and 100 μL of
combined solution is withdrawn and diluted with 900 μL of deionised (DI) water.
2. Diluted solution is pipetted up and down to ensure homogeneity of mixture and
1 mL of it is transferred into 1 x 1.7 ml quartz cuvette.
3. 1 mL of DI water is transferred into another 1 x 1.7 ml quartz cuvette as a blank
test.
Optical density measurement
1. Spectrophotometer is set to wavelength of 260 nm.
4
 2. DI water cuvette is inserted into the spectrophotometer, clear side facing the
user, and used to zero the machine.
3. Plasmid solution is then inserted, clear side facing the user, and the reading
of A260 is taken.
4. Spectrophotometer is set to wavelength of 280 nm, and repeating step 2 and
3, A280 is taken.
4. Results
Based on spectrophotometer,
A260 = 0.065 A
A280 = 0.045 A
Hence,
𝐴260 0.065
= = 1.444
𝐴280 0.045
For double stranded DNA, 1 A260 = 50 µg/ ml.
Hence,
Concentration of DNA extracted = 50 x 0.065 = 3.25 µg/ ml.
5. Discussion
As mentioned above, a good A260/A280 ratio should be between 1.8 – 2.0. However,
the ratio achieved fell short, at 1.44.
A260 is the optical density of the solution, after UV light is passed through the quartz
cuvette with absorption from nucleic acid, while A280 is the optical density of the
solution, after UV light is passed through the quartz cuvette after absorption from
protein. High values of A280 will bring the ratio lower, while low values of A260 will also
bring the ratio lower. Hence, two possible, non-mutually exclusive conclusion can be
drawn:
1. Concentration of nucleic acid is low, hence giving a low A260 value and
2. Concentration of proteins is high, hence giving a high A280 value.
This section will discuss possible factors that resulted in low DNA extraction yield.
5.1. Lysis reaction not allowing to react fully
The addition of Buffer P2, SDS-based lysis buffer with RNase, is to break down the
phospholipid bilayer and to denature the chromosomal DNA, plasmids and proteins,
due to the alkaline nature. RNase cleaves the RNA present in the cell. However, this
reaction takes time to occur, but also must be ensured it does not proceed to
completion. Hence, it was advised for the reaction to proceed no longer than 5
minutes, before neutralisation with Buffer N3, which helps to renature the plasmid
DNA but not the chromosomal DNA.

5
Buffer N3 was added into the cell pallet around the 3 minutes mark. This could explain
why A280 value is high, as there could be more proteins that was denatured by Buffer
P2.

However, it should be noted A260 value is based on the concentration of nucleic acid,
but it does not distinguish between RNA and DNA. RNase reaction with RNA present
in the cell might not have completed totally by the time Buffer P2 was added, but A260
value would have been higher if it were the case. Hence, the possibility of lysis reaction
being the main reason for low DNA yield might not be the main issue, but a possible
factor.
5.2. Transfer of plasmid solution to QIAprep column.
After the centrifugation of solution after adding Buffer N3, the solution is observed to
separate into the supernatant (solution with plasmid), and a white precipitate. The
supernatant is pipetted into the QIAprep column, with emphasis to not touch the
particulate.
The white precipitate was formed by the addition of Buffer N3, a high salt potassium
acetate buffer. Denatured DNA, due to its molecular weight, binds to the potassium
and SDS from Buffer P2, as well as the denatured protein (Ehrt & Schnappinger,
2003).
One possible error is during the transfer of supernatant, pipette tip grazed the white
precipitate and the contaminants, namely denatured protein, was transferred over to
the QIAprep column. Hence, there could be trace amount of protein present in the
solution after elution. This gives a high A280 value as there is protein contaminant
present that was transferred from the white precipitate, lowering the A 260/A280 ratio.
5.3. Elution waiting time
Buffer EB, an elution buffer, is added to elute the materials from the silica resin
column. It should be noted that after the addition of Buffer EB, there should be a
settlement time of 1 minute, followed by a centrifuge of 1 minute.
One possible error from this experiment was that the 1 minute of settling time was
overlooked in the procedure, and hence was skipped. This may have resulted in an
incomplete elution from the column, and therefore lowering the DNA concentration
and thereafter A260 value being lower.
Therefore, a combination of these three factors could have contributed to a lower-than-
expected A260/A280 ratio.
6. Conclusion
In summary, plasmid isolation achieved in this experiment was of below quality. Three
possible reasons that affected the yield of DNA could be that the lysis reaction did not
complete totally, contamination with protein during transfer and the elution waiting
period not being carried out.

6
7. Logsheet
Name: Brandon Lam Chi Ee
Student ID: U2220508G
Group: CBE 02/ Group 3
Date: 31th August 2022
Dilution Factor: 10
What is the A260: 0.065 A
What is your plasmid DNA concentration: What is your A280: 0.045 A
What is your A260 / A280: 1.444
Do you achieve good plasmid DNA isolation? No
What is the rationale behind your answer? It is not within the range of 1.8 – 2.0 of
plasmid DNA isolation, which is used as a benchmark.
Please indicate step(s) that might cause contamination of your protein purification.
1. Buffer P2 and reaction time and
2. Contamination of white precipitate during transfer and
3. Did not wait for elution to carry out.

7
8. References
Cronan, J. E. (2001). Escherichia coli as an Experimental Organism. eLS: John
Wiley & Sons. Retrieved 5 September, 2022, from
https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470015902.a0002026.pu
b2
Ehrt, S., & Schnappinger, D. (2003). Isolation of Plasmids from E. coli by Alkaline
Lysis. In N. Casali, A. Preston, N. Casali, & A. Preston (Eds.), E. coli Plasmid
Vectors (Vol. 235, p. 75). Totowa, New Jersey: Humana Press.
G Bio Sciences. (n.d.). Plasmid Isolation. Retrieved 5 September, 2022, from
Gbiosciences.com: https://cdn.gbiosciences.com/pdfs/protocol/BE-
310_protocol.pdf
Jeltsch, M. (26 September, 2016). Plasmid with insert. Retrieved 5 September, 2022,
from Wikipedia: https://commons.wikimedia.org/w/index.php?curid=51664406
QIAGEN. (n.d.). QIAprep Spin Miniprep Columns. Retrieved 5 September, 2022,
from QIAGEN.com: https://www.qiagen.com/us/products/discovery-and-
translational-research/lab-essentials/plastics/qiaprep-spin-miniprep-columns/
Science Learning Hub. (25 March, 2014). E. coli – the biotech bacterium. Retrieved
5 September, 2022, from E. coli – the biotech bacterium — Science Learning
Hub: https://www.sciencelearn.org.nz/resources/1899-e-coli-the-biotech-
bacterium
World Health Organization. (7 February, 2018). E. coli detail. Retrieved 5
September, 2022, from World Health Organization news room:
https://www.who.int/news-room/fact-sheets/detail/e-coli