Lab Report 2

Radita Khan

ID:22336021

Submitted for: BTE355

Biotechnology Lab IV

Section:05

Submitted to:

Tushar Ahmed Shishir

Date:13.11.24

Brac University

Fall Semester 2024

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Experiment – 02
Name of the experiment: Isolation of Plasmid DNA from Escherichia coli

Plasmids are predominantly used as vectors in genetic engineering due to their ability to carry
foreign genes, which allows for the creation of recombinant DNA. They possess an origin of
replication (ori) that enables independent replication within host cells, ensuring multiple copies
of the plasmid can be produced. Additionally, plasmids often include selectable marker genes,
such as antibiotic resistance, facilitating the identification of successfully transformed cells. They
feature multiple cloning sites (MCS) for easy insertion of DNA fragments and are small in size,
making them easier to manipulate in the lab. Their rapid replication in host cells allows for
large-scale production of desired products, and their natural occurrence in bacteria enhances their
compatibility and effectiveness as vectors. These characteristics collectively make plasmids
invaluable tools in molecular biology and biotechnology.

Plasmid isolation leverages the fact that plasmids are relatively small, supercoiled (closed
circular) DNA molecules, while bacterial chromosomal DNA is significantly larger and less
supercoiled. This difference in structure facilitates the selective precipitation of chromosomal
DNA and cellular proteins, allowing for the purification of plasmids and RNA molecules.
Following gentle lysis of the cells, all intracellular macromolecules are removed, enriching and
purifying the plasmid DNA. Smaller plasmids are generally easier to isolate intact.

For effective plasmid isolation, bacterial cultures should be grown to the late logarithmic or early
stationary phase, which typically requires overnight cultivation under optimal conditions to
achieve a robust culture. It is crucial to completely remove the growth media from the cell pellets
after centrifugation during harvesting.
Cells are lysed under alkaline conditions that denature both nucleic acids and proteins. Upon
neutralization with potassium acetate, chromosomal DNA and proteins precipitate because they
cannot renature properly due to their large size. In contrast, plasmids can renature correctly and
remain in solution, effectively separating them from chromosomal DNA and proteins.
Tris buffer is commonly used for DNA preparations because it maintains a slightly alkaline pH
(7.5-8.2), which is ideal for storing DNA. EDTA is also a key component in plasmid preparations
as it inhibits nuclease activity. For long-term storage, plasmid DNA should be frozen in aliquots
of TE buffer.

DNA is highly sensitive to mechanical stress; therefore, it is essential to avoid shearing forces
from vigorous mixing or rapid pipetting immediately after cell lysis. All mixing steps during and
following lysis should be conducted gently by inverting the tubes several times (8-10 times).
Additionally, gloves should be worn to prevent contamination with DNases, and autoclaved
(DNase-free) buffer solutions, tubes, and pipette tips should be utilized.

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Media, Buffers and Other Requirements:
1. Active culture of E. coli DH5α containing pGLO Plasmid in Ampicillin (+) LBmedium
Alkaline lysis solutions:
Solution I (1M Tris-HCl pH-8.0, 0.5M EDTA, 1M glucose in distilled water), pH-8.0
Solution II (10 N NaOH, 1% SDS in distilled water).
Solution III (5M Sodium Acetate pH-5.2, glacial acetic acid in distilled water), pH-5.2
2. Ethanol.
3. phenol: chloroform: IAA (25:24:1, v/v)
4. chloroform: IAA (24:1)
5. TE Buffer (pH8.0)
Procedure:
1. Bacterial cells harboring the desired plasmid were grown overnight in 25-50 ml of LB medium
containing the appropriate antibiotic.
2. A 1.0 ml aliquot of cells was transferred and harvested by centrifugation at 10,000 RPM for 3
minutes (twice if needed).
3. The pellet was then resuspended in Solution 1 (200 µl).
4. To this freshly prepared solution, Solution 2 (400 µl) was added and mixed by gentle
pipetting.
5. The cells were incubated at room temperature for 5 minutes for cell lysis.
6. Ice-cold Solution 3 (300 µl) was added to this, mixed by inversion, and incubated on ice for 10
minutes.
7. The mixture was centrifuged at 12,000 RPM for 10 minutes.
8. The supernatant was collected in a fresh tube.
9. An equal volume (600 µl) of phenol: chloroform: isoamyl alcohol (25:24:1) was added to this,
mixed by inversion (for a minimum of 5 minutes), and centrifuged at 12,000 RPM for 5 minutes.
10. The supernatant was carefully collected, and an equal volume of chloroform: isoamyl alcohol
(24:1) was added, mixed by inversion (for a minimum of 5 minutes), and centrifuged at 12,000
RPM for 5 minutes.
11. The supernatant was collected, and then 2.5 times the volume (750 µl) of isopropanol was
added to this and mixed by inversion.
12. The mixture was incubated in a -20°C freezer for a minimum of 20 minutes.
13. After that, centrifugation was performed at 12,000 RPM for 15 minutes.
14. The DNA pellet was washed in 70% ethanol, air-dried, and suspended in 10-15 µl of TE
buffer.
15. The plasmid was stored at -20°C in the freezer.

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Observation:

Fig 2.1 : Layers formed after adding PCI.

Fig 2.2: Pellet formed after adding isopropanol and keeping in the freezer at -20 degree celsius
for 20 minutes.

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Result :
White Pellet was seen after adding isopropanol and keeping in the freezer at -20 degree celsius
for 20 minutes.

Discussion :
During the plasmid DNA isolation procedure, a significant issue arose due to the delayed
incubation time after the addition of Solution II (10 N NaOH, 1% SDS). The protocol specified a
5-minute incubation period; however, the actual timing began only after the last group completed
adding Solution II, resulting in an extended waiting period of approximately 8 minutes for some
groups.

This delay had a notable impact on the quality of the isolated DNA. Prolonged exposure to
alkaline conditions can lead to genomic DNA degradation, which is particularly concerning
when isolating plasmid DNA. The degradation may result in lower yields and compromised
integrity of the plasmid DNA, ultimately affecting downstream applications such as cloning or
sequencing.

The presence of a larger-than-expected white pellet after centrifugation raised concerns about the
purity of the isolated plasmid DNA. Typically, plasmid DNA yields a very small pellet due to its
size and quantity, often making it visually indistinct. In this case, however, the visible pellet was
larger than anticipated, indicating potential contamination with genomic DNA and salts. This
observation suggested that genomic DNA may not have been fully precipitated during the
neutralization step or that residual salts contributed to the excess mass.

To mitigate these issues in future experiments, it is essential to ensure strict adherence to timing
protocols. Synchronizing actions among groups will prevent delays that could compromise DNA
integrity. Additionally, implementing a more rigorous monitoring system for timing could help
maintain consistency across all groups.

Optimizing the lysis and neutralization steps could further improve results. For example,
reducing the incubation time with Solution II could minimize genomic DNA degradation while
still allowing sufficient lysis of bacterial cells. By addressing these factors, we can enhance the
purity and yield of plasmid DNA in future experiments and ensure high-quality results free from
genomic contamination.

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Precautions:
1. Perform all steps quickly to prevent degradation of plasmid DNA.
2. Use gentle mixing to avoid breaking the DNA.
3.Wear appropriate personal protective equipment (gloves and goggles) when handling
chemicals.
4. Ensure all reagents and materials are sterile and DNase-free to prevent contamination.
5. Keep samples and reagents cold to maintain DNA integrity.
6. Carefully handle centrifugation steps to avoid disturbing pellets.
7. Add neutralizing solutions promptly after lysis to minimize exposure to harsh conditions.
8. Use the correct volumes of reagents for effective precipitation.
9. Wash DNA pellets thoroughly to remove contaminants.
10. Minimize mechanical stress during mixing and handling.
11. Consider using RNase to eliminate RNA contaminants.
12. Store plasmid DNA properly to prevent degradation over time.