Lab Report 3

Radita Khan

ID:22336021

Submitted for: BTE258

Biotechnology Lab III

Section:05

Submitted to:

MD Tausif Ur Rashid

Date:15.09.24

Brac University

Summer Semester 2024

Name of Experiment : Extraction of Chloroplast DNA from leaf samples by Miniprep
Protocol.
Abstract :
This experiment focused on the isolation of cpDNA from leaf samples using a Miniprep
protocol. The Miniprep method is fast and efficient for CPDNA extraction, crucial in research
studies within plant genetics and molecular biology. Freshly collected leaves underwent
disruption of cell walls and membranes and then underwent differential centrifugation to separate
the chloroplasts. The DNA extraction process consists of the breaking open of cells, unfolding
proteins, and precipitation. Later on, the purified cpDNA was quantified and checked for quality.
The results show that the Miniprep method yields high-quality chloroplast DNA suitable for
PCR amplification and sequencing procedures.

Principle :

The primary distinction between CTAB isolation and cpDNA isolation lies in their objectives
and outcomes. While the CTAB method is designed for total DNA extraction, cpDNA isolation
aims to obtain a high-purity chloroplast DNA sample. Consequently, the CTAB method may
result in significant contamination from organellar DNA, while cpDNA isolation methods are
optimized to minimize such contamination. Researchers must choose the appropriate method
based on their specific experimental requirements, balancing the need for total genomic DNA
versus the need for high-purity cpDNA. Now let’s come to the reagents that re being used :

1. CTAB Lysis Buffer

CTAB (cetyltrimethylammonium bromide) lysis buffer is used to disrupt cell membranes and
solubilize nucleic acids while precipitating polysaccharides and proteins. This buffer is
particularly effective in extracting high molecular weight DNA, including cpDNA, from plant
tissues.

2. Sorbitol
Sorbitol is included in cpDNA isolation protocols to help maintain osmotic balance during the
extraction process. It protects chloroplasts from osmotic shock and helps preserve their integrity
during cell lysis. By stabilizing the chloroplast membranes, sorbitol enhances the yield of intact
chloroplasts, which is critical for obtaining high-quality cpDNA.

3. Tris-HCl
Tris-HCl is a buffering agent that maintains a stable pH during the extraction process. It is
essential for preserving the integrity of nucleic acids, as nucleic acids are more stable in neutral
to slightly alkaline conditions (pH 7.0-9.0). This stability helps prevent hydrolysis and
degradation of the cpDNA during extraction.

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4. EDTA
EDTA (ethylenediaminetetraacetic acid) is a chelating agent that binds divalent metal ions, such
as Mg²⁺ and Ca²⁺. By sequestering these ions, EDTA inhibits the activity of nucleases that could
degrade DNA, thus protecting the cpDNA during the extraction process.

5. NaCl
Sodium chloride (NaCl) is used to create a high-salt environment that helps reduce electrostatic
interactions between nucleic acids and proteins. This reduction minimizes the adsorption of
nuclear DNA (nDNA) to the chloroplast membranes, thereby decreasing contamination and
improving the purity of the isolated cpDNA. Additionally, NaCl aids in the precipitation of
proteins and polysaccharides, which are common contaminants in plant tissues.

6. Magnesium Chloride (MgCl₂)

Magnesium chloride (MgCl₂) serves as a cofactor for various enzymes involved in DNA
replication and transcription. It stabilizes the structure of the DNA double helix and is essential
for the proper functioning of DNA polymerases during any subsequent amplification or analysis
of the extracted cpDNA.

7. Sodium Dodecyl Sulfate (SDS)

Sodium dodecyl sulfate (SDS) is an anionic detergent that lyses chloroplast membranes,
facilitating the release of cpDNA. SDS also denatures proteins, aiding in the removal of protein
contaminants during the extraction process. By binding to proteins and polysaccharides, SDS
enhances the solubility of nucleic acids and helps precipitate contaminants.

8. Phenol Chloroform and Isoamyl Alcohol ( PCI)

Phenol is used primarily for its ability to denature proteins. When mixed with an aqueous
solution containing DNA, phenol helps to precipitate proteins at the interface between the
aqueous and organic phases, allowing for the separation of DNA, which remains in the aqueous
layer. Additionally, phenol inactivates nucleases that could degrade DNA, protecting the integrity
of the extracted cpDNA. The pH of the phenol solution is typically controlled (using Tris- or
TE-saturated phenol with a pH around 7.8) to suppress the partitioning of DNA into the organic
phase.

Chloroform and isoamyl alcohol are used in the extraction phase to separate nucleic acids from
proteins and other contaminants. Chloroform increases the density of the organic phase,

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preventing phase inversion and aiding in protein denaturation. Isoamyl alcohol acts as an
anti-foaming agent, reducing foaming and emulsification during the extraction process.

9. Centrifugation
After the addition of phenol, chloroform, and isoamyl alcohol, the mixture is centrifuged to
separate the phases. The aqueous phase contains the DNA, while the organic phase contains
denatured proteins, lipids, and phenol. The interphase contains partially denatured proteins and
cellular debris.

10. Isopropanol
Isopropanol is used to precipitate DNA from the aqueous phase. DNA is less soluble in
isopropanol than in water, causing it to aggregate and form a visible precipitate upon
centrifugation. Isopropanol is often more effective than ethanol for precipitating DNA, especially
at low concentrations, and requires a smaller volume for precipitation.

11. TE Buffer
TE buffer (Tris-EDTA) is commonly used for the storage of isolated DNA. It provides a stable
pH and protects DNA from degradation by chelating divalent metal ions that could activate
nucleases. TE buffer is typically used after cpDNA extraction to resuspend and store the purified
DNA.

12. 70% Ethanol

70% ethanol is used for washing the DNA pellet after precipitation. It helps to wash away salts
and other impurities while allowing the cpDNA to remain in a soluble form. The use of ethanol
also aids in the final purification step, ensuring that the isolated cpDNA is of high quality and
suitable for downstream applications.

Procedures :

1.A clean and fresh leaf sample was cleaned and dried to remove all surface contaminants.
2. The dried leaf was ground using a mortar and pestle into a very fine paste, while being very
careful not to rupture any of the cell structures and release the chloroplasts.
3. Aided the lysis of cell membranes and release of DNA, to the leaf powder sample, 500 ul of
DNA extraction buffer was added to form a homogenous paste.
4. The thus-formed paste was filled in a microcentrifuge tube to a volume of 0.5 ml.
5. The tube was then incubated in a water bath at a temperature of 65°C for 20 minutes. During
incubation, to avoid hot spots, the tube was inverted after ten minutes to ensure even heating.
This helped break down the cellular components and freed the cpDNA.
6. After incubation, 700 ul of chloroform:alcohol mixture (24 :1) was added to the tube. This
mixture denatures the proteins and separates the DNA into aqueous phase.

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7. Then, the sample mixture was inverted gently for ten minutes to achieve adequate phase
separation and proper mixing.
8. Centrifugation at 11,000 rpm for eight minutes separated the lower organic phase, the
interphase containing the denatured protein, and the upper aqueous phase carrying cDNA.
9.With extreme caution, 500 ul of the upper aqueous phase were moved with a micropipette to a
fresh microfuge tube in order to prevent contamination from the organic phase and interphase.
10. To precipitate the CPDNA, the separated aqueous phase was combined with 700 ul of
isopropanol.
11. The mixture was given three gentle stirs to make sure the isopropanol was distributed evenly.
12. To promote DNA precipitation, the tube was then incubated for 20 minutes at -20°C in an ice
bath.
13. To pellet the cpDNA, the mixture was centrifuged at 13,000 rpm for 12 minutes following
the ice bath incubation.
14. The DNA pellet was placed at the bottom of the tube and the supernatant was disposed of
properly.
15. To get rid of any last bits of salt and contaminants, the DNA pellet was washed in 500 ul of
ice-cold ethanol.
16. To re-pellet the DNA, the tube was centrifuged twice for two minutes at 8,000 rpm.
17. The DNA pellet was then air-dried briefly after careful removal of the ethanol to allow
evaporation of any residual ethanol.
18. The pellet was resuspended in 20 – 50 ul TE buffer that stabilizes and protects DNA from
degradation. 19 points.
19. The resultant solution of reconstituted CPNA was stored at -20°C for a short period of time
until needed.

Reagents:
1.Isolation buffer (0.33M sorbitol, 0.1M Tris-HCl pH 7.8, 5mM MgCh, 10mM NaCI, 2mM
EDTA.)
2.Lysis buffer (2% CTAB, 100 mM Tris-HCl p 8.0,50 mM EDTA pH 8,0, 1.4 M NaCl)
3.TE buffer (10 mM Tris-HCI pH 8.0, 1 mM EDTA pH 8.0)
4.Phenol: Chloroform: isoamyl alcohol (25:24:1)
5. Isopropanol
6. Ethanol (70%)

Materials:
1. Plant material (fresh leaves, needles, etc.)
2. Mortar and pestle / blender
3. Centrifuge tubes
4. Miracloth / filter paper
5. Centrifuge

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Result and Observations :

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Discussion

In the experiment the attempt to isolate chloroplast DNA (cpDNA) was unsuccessful in yielding
purified cpDNA, primarily due to the omission of several critical reagents and the improper
centrifugation conditions employed during the process. Sodium sulfite was not included in the
extraction protocol, which serves as an important antioxidant that prevents the oxidation of
phenolic compounds that can interfere with DNA quality; without it, the extracted cpDNA may
have been compromised by oxidized contaminants that could inhibit downstream applications.
The absence of DNA Extraction Buffer (DEB), which typically contains essential components
such as Tris, EDTA, and salt, is another significant oversight, as DEB plays a crucial role in
stabilizing nucleic acids and inhibiting nucleases that can degrade DNA; the lack of DEB likely
allowed nucleases to remain active during the extraction, resulting in degradation of the cpDNA
and a lower yield. Sorbitol, which helps maintain osmotic balance and protects chloroplasts from
osmotic shock during cell lysis, was also omitted, potentially compromising the integrity of the
chloroplasts and leading to reduced recovery of intact cpDNA. While chloroform and isoamyl
alcohol were used in the extraction process, the failure to use the complete
phenol-chloroform-isoamyl alcohol (PCI) mixture complicated the extraction; phenol is
particularly important because it acts as a potent protein denaturant, effectively disrupting the
structure of proteins and causing them to precipitate at the interface between the aqueous and
organic phases. This separation is essential for isolating high-quality nucleic acids, as the polar
nucleic acids remain in the aqueous phase while denatured proteins and other contaminants
dissolve in the phenol phase. The omission of phenol means that proteins would not be
adequately removed, leading to contamination of the cpDNA with residual proteins and
nucleases that could degrade the DNA. The initial centrifugation step was performed at an
insufficient speed of 500 rpm, which is inadequate for effectively separating chloroplasts from
cellular debris; this step is crucial for enriching the chloroplast fraction, and without it, the
cpDNA could not be isolated effectively. Higher centrifugation speeds (e.g., 10,000 RPM or
higher) are necessary to pellet the chloroplasts and achieve a clear separation of the cpDNA from
other cellular components. Although TE buffer was used in the process, it is essential to ensure
that it is applied as the final step for resuspending the isolated cpDNA, as it provides a stable pH
environment and chelates divalent metal ions that could activate nucleases, thus protecting the
DNA from degradation. In conclusion, to improve the cpDNA isolation process, it is essential to
incorporate all the critical reagents, including sodium sulfite, DEB, sorbitol, phenol, chloroform,
isoamyl alcohol, and to ensure that TE buffer is used as the last step. Additionally, performing
the initial centrifugation step at the appropriate speed is crucial for effective separation and
purification of cpDNA. By following these recommendations, future experiments are likely to
yield higher quality and quantity of isolated cpDNA, suitable for downstream applications such
as PCR, cloning, or sequencing.

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Precautions:
1.To avoid powder getting stuck in the imperfect grooves, use caution when using the mortar and
pestle.
2.When using an anti foaming chemical, the cuvette must remain upright to avoid foaming.
3. When using a centrifuge, equilibrium must be maintained.
4.In a water bath, the cuvette should be partially submerged to avoid tipping over.