LANZUELA, MILLICENT BERNADETTE S.

BIO 101 – Z C2
Plant DNA Isolation
MAJOR PROCEDURES
- The preparation for plant DNA isolation is determined by the species, type of tissue or
sample available and the analysis required on the DNA.
- Interfering substances such as polyphenols, polysaccharides and secondary metabolites
contained in plants affect the extraction of genomic DNA and inhibit reactions such as the
polymerase chain reaction (PCR).
DNA protocols are crucial and should be efficient, yet cost-effective, in the removal of
these contaminants to obtain a good quality DNA suitable for further experimentation. In the
study of Pathak, A. et. al. (2013) in DNA isolation from fresh leaf tissue of Tylophora indica and
Bacopa monnieri, the DNA protocol used was the Doyle and Doyle’s method with certain
modifications in the procedure.
 Fresh tissue is usually preferred in most cases of DNA extraction. It may be
extracted immediately or stored for hours of day at a condition that will not
allow growth of fungi such as refrigeration or preservation by alcohol (Henry,
R.J. 2001).
 Plant samples can also be collected from dry tissue, and pollen or from
ancient DNA, bark and plant pathogens which are more difficult to sample.
1. 5 mL CTAB isolation buffer was preheated in a centrifuge tube (15 mL) at 65 °C for
15 min and was added to fresh healthy leaves (1 g) along with 2-mercaptoethanol
(0.2%).
 Buffers control the pH of the extraction solution. Other components include
salts, chelating agents, detergents, etc.
2. The leaves were ground into a paste, put into the centrifuge tube (15 mL) and kept in
a water bath at 65 °C for 1.5 h.
 Tissue disruption may be done through physical disruption where minimum
mechanical disruption is done to release the DNA from the tissue, whereas,
surplus action might shear the DNA (Henry, R.J. 2001). Heating at high
temperature and microwave heat can also be utilized.
3. The obtained paste was distributed equally into Eppendorf tube and an equal volume
of chloroform / isoamylalcohol (24:1) was added, mixed well and centrifuged at 6000
rpm for 15 min at 15 °C.
4. The aqueous phase was collected and carefully transferred to a new Eppendorf tube
using a wide bore tip.
5. Equal volume of cold isopropanol was added, well mixed and kept overnight at –20
°C. On the next day, it was centrifuged at 12 000 rpm for 15 min at 15 °C.
Supernatant was discarded and 500 μL of ethanol (85%) was added.
 6. The pellet formed was mixed by gentle tapping and centrifuged at 10 000 rpm for 10
min at 15 °C. 500 μL of 75% ethanol was added and centrifuged again and the
supernatant was discarded.
7. The pellet was allowed to air dry for 45 min to 1 h.
8. The pellet was resuspended in 30 μl TE buffer (1X) and the mixture was kept in a hot
air oven (65 °C) for 10 min.
9. It was then cooled at room temperature and kept in refrigerator at 4 °C.
10. After the DNA as isolated, various methods were used to check its quality and
quantity.
 Quantification of the DNA isolated can be done through spectrophotometric
analysis which can be determined by taking the absorbance at 260 nm (1 O.D.
= 50 μg mL–1 of double stranded DNA).
 Agarose gel electrophoresis, 0.8% agarose gel was prepared and placed into
an electrophoretic unit with 1X TAE buffer. This allows estimation of the
quality and quantity of the isolated DNA and more accurate than UV-based
methods (Henry, R.J. 2001). Ethidium bromide, as the staining gel, was used
to observe the intact band of genomic DNA.
 Purity of the DNA can be estimated by absorbance wherein A 260/A280 = 1.8
(Henry, R.J. 2001) and impurities such as polysaccharides, proteins and
polyphenolics are determined at A260/280, A260/230 and A260/270 (Pathak, A. et. al.
2013).
 Amplification test as qualitative was conducted to test for contaminants that
could hinder PCR reaction.
o The amplified products were examined by electrophoresis on 1.2%
agarose gel stained with ethidium bromide. Electrophoresis is used to
assess the size of DNA molecules in extracts.
REFERENCES
Henry, R. J. 2001. Plant Genotyping: the DNA Fingerprinting of Plants. Plant DNA extraction.
CAB International. DOI: 10.1079/9780851995151.0239
Pathak, A. Dwivedi, M. Laddha, N. Begum, R. Joshi, A. (2013). DNA isolation from fresh leaf
tissue of Tylophora indica and Bacopa monnieri. Environmental and Experimental
Biology. 11: 69–71
Animal DNA Isolation
MAJOR PROCEDURES
Procedure 1
1. Centrifuge 10 mL of raw milk at 7000 g for 10 minutes (4° C), remove the milk fat and
most of the supernatant from above somatic cells and milk proteins pellet, and transfer
the pellet with the remainder of the supernatant to a 2 mL sterile tube. Centrifuge the
mixture at 5000 x g for 3 minutes (4 °C) and remove the supernatant.
2. Wash the pellet with 1 mL of buffer. Centrifuge this mixture at 5000 x g for 3 minutes
(4° C), remove the supernatant, and repeat this step until clear supernatant is obtained.
3. Add 1 mL of lysis buffer to the pellet and incubate this mixture at 65° C for
approximately 20–30 minutes. After this time, the strings of DNA clumps will be visible
in the liquid.
4. Attach the strings of DNA clumps to the wall of a new sterile 1.5 mL tube by pipette.
Then, discard leftover supernatant that has dropped to the bottom and wash DNA twice
with 100 μL of 70% ethanol. Centrifuge the mixture at 10000 x g for 1 minute at room
temperature and discard the supernatant.
5. Dissolve the DNA pellet in 50 – 100 μL of deionized water or TE buffer.
Procedure 2
1. The same step is followed from step 1 to 2 of Procedure 1.
2. Add 1 mL of lysis to the pellet and incubate this mixture at 65° C for approximately 60–
90 minutes.
3. Cool down the mixture to room temperature and add 450 μL of protein precipitation
buffer, vortex, and then centrifuge the mixture at 16000 x g for 8 minutes (10° C).
4. Transfer the supernatant to a new tube and add 600 μL of 100% isopropanol. Centrifuge
the mixture at 10000 x g for 8 minutes and remove the supernatant.
5. Wash the DNA pellet twice with 70% ethanol and air dry.
6. Dissolve the DNA pellet in 50-100 μL of deionized water or TE buffer.
REAGENTS/CHEMICALS USED
Genomic Plant DNA isolation and its analysis
 CTAB isolation buffer
o 2% CTAB,
o 1.4 M NaCl - influence the solubility of DNA and other molecules present in the
extraction.
o 0.2% 2-mercaptoethanol
o 20 mM EDTA
o 100 mM Tris HCl
 chloroform:isoamylalcohol (24:1) – prevent frothing and esterification of lipid.
o Chloroform improves efficiency of nucleic acid extraction by denaturing protein,
aids in the dissociation of protein from nucleic acid apart from catalysis of
removal of lipid.
o When present in large concentrations, they cause dehydration of DNA by
extracting water.
 isopropanol and ethanol (85 and 75%) – aside from precipitating DNA, it displaces water
of hydration required for solubilization of DNA creating a hydrophobic environment.
 1X Tris EDTA buffer
o 10 mM Tris HCl
o 1 mM EDTA, pH 7.4
 50X Tris acetic acid EDTA buffer
o 24.2 g Tris base
o 5.71 mL glacial acetic acid
o 0.5 M EDTA, pH 8.0
 agarose
 ethidium bromide (EtBr)
 Bromophenol blue.
Animal DNA Isolation from Milk Somatic Cells
 Buffer
o 15 mM Tris – HCl (pH 7.4 - 7.6)
o 25 mM NaCl
o 5 mM MgCl2
o 15 mM Na2HPO4
o 2.5 mM EDTA – a chelating agent that binds metal ions in the extraction solution.
o 1% sucrose
 Lysis buffer
o pH ¼ 8.8
 o 6% SDS – a detergent that aids in disruption of phospholipid bilayer of cell
membrane and nuclear membrane.
o 3 mM MgCl2
o 15 mM Tris-HCl
o 0.5% DMSO
o 6% acetone
 70% ethanol aside from precipitating DNA, it displaces water of hydration required for
solubilization of DNA creating a hydrophobic environment.
 50 – 100 μL of deionized water – solvent used
 TE buffer (pH 8.0)
o 10 mM Tris
o 1 mM EDTA
 450 μL protein precipitation buffer (pH 5.0)
o 2.35 M NH4Cl
o 1.15 M NaCl
o 38% ethanol
 600 μL 100% isopropanol