M.Sc.

Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

ISOLATION OF GENOMIC DNA

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AIM:
To extract genomic DNA from bacteria using CTAB/NaCl method
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INTRODUCTION
Genomic DNA is the chromosomal DNA. All organisms contain the same genomic DNA in
every cell; however, only certain genes are active in each cell to allow for cell function and
differentiation. In prokaryotes, DNA is double stranded and circular and is found throughout the
cytoplasm and is called nucleoid. In eukaryotes, DNA is located in the nucleus and in mitochondria
or chloroplasts. The DNA in the nucleus is double stranded and linear, whereas the DNA in
mitochondria and chloroplasts is like prokaryotic DNA, double stranded and circular. The DNA in
prokaryotes is relatively free of associated protein, but the DNA in the nucleus of eukaryotes is
associated with basic proteins, called histones.
The isolation of DNA is one of the most commonly used procedures in many areas of
bacterial genetics, molecular biology and biochemistry. Purified DNA is required for many
applications such as studying DNA structure and chemistry, examining DNA protein interactions,
carrying out DNA hybridizations, sequencing or PCR, performing various genetic studies or gene
cloning. High purity DNA is ideal for various gene manipulation experiments
Many different techniques are available for isolating genomic DNA from prokaryotic cells;
however, the choice of method depends on degree of purity of DNA required for the analysis to be
performed. Some DNA analyses (i.e those using restriction enzymes) require DNA of high purity
and relatively large amounts. However, analyses based on PCR only require very small amounts of
DNA whose quality can be crude. The bacterium replicates its DNA in favourable conditions of
nutrition, pH and temperature. The process of bacterial cell division is much simpler than
eukaryotic cells, and hence, bacteria are able to grow and divide much faster.
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PRINCIPLE
The isolation of DNA from bacteria is a relatively simple process. The organism to be used
should be grown in a favorable medium at an optimal temperature, and should be harvested in
late log to early stationary phase for maximum yield.
There are many methods for isolating DNA; however, all follow the common steps of cell
breaking, protein removal followed by release of the genetic material. Cell walls and membrane
disruptions usually are accomplished with an appropriate combination of enzymes to digest the
cell wall (usually lysozyme) and detergents to disrupt membranes. Most common ionic detergent
used in this step is sodium dodecyl sulphate (SDS). When the cell is disrupted, the nucleases can
cause extensive hydrolysis of DNA. Nucleases apparently present on human fingertips are
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M.Sc. Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

notorious for causing spurious degradation of nucleic acids during purification. Chelating agents
are added to remove metal ions required for nuclease activity. RNA is usually degraded by the
addition of DNase free RNase. Proteins are subjected to chemical denaturation and/or enzymatic
degradation by addition of proteinase-K. The most common technique of protein removal involves
denaturation and extraction into organic phase viz. phenol and chloroform. The DNA in the
aqueous phase is precipitated with ethanol or isoamyl alcohol. The purified DNA is then
resuspended and stored in TE buffer or sterile distilled water. The quality of the DNA can be
checked by agarose gel electrophoresis and spectrophotometer.
EQUIPMENTS AND MATERIALS REQUIRED
1. High speed centrifuge; 2. Microfuge; 3. Auto pipettes- 2-20μL; 20-200 μL; 200-1000 μL
4. Waterbath; 5. -200 C Deep freezer; 6. Refrigerator; 7. Container of ice; 8. Sterile microcentrifuge
tubes; 9. Sterile pipet tips; 10. Sterile Pasteur pipets
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REAGENTS REQUIRED AND THEIR ROLE
1. Luria–Bertani Broth:
Luria–Bertani (LB) broth is a rich medium that permits the fast growth and better yields for
many species including Escherichia coli. Easy to make, fast growth of the most E. coli strains,
readily available and simple compositions. E. coli grow to optical density (OD)600 2–3 in LB broth
under normal shaking incubation conditions in 24 h.
2. Tris EDTA (TE) Buffer:
As a major constituent of Tris EDTA (TE) buffer, Tris acts as a common pH buffer to control
pH, while EDTA chelates cations like Mg2+. Thus, TE buffer is helpful to solubilise DNA and protect
it from degradation.
3. Sodium Dodecyl Sulphate (SDS):
SDS is a strong anionic detergent that can solubilise the membrane proteins and lipids. This
will help the cell membranes to break down and expose the chromosomes to release DNA.
4. Proteinase-K:
Proteinase-K at 20 mg/ml is a very good enzyme that degrades most types of protein
impurities to get a quality DNA product. It is also responsible for the inactivation of nucleases, thus
preventing damage of isolated DNA.
5. NaCl Solution:
5 M NaCl provides Na+ ions that block negative charge of phosphates of DNA. Negatively
charged phosphate in DNA causes molecules to repel each other. The Na + ions form an ionic bond
with the negatively charged phosphates; thus, neutralise the negative charges and allowing the
DNA molecules to come together.
6. Cetyl Trimethyl Ammonium Bromide (CTAB):

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M.Sc. Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

CTAB is a detergent that helps to lyse the cell membrane. Apart from that, CTAB-NaCl
solution binds with proteins in the digested cell lysate and helps in separation of DNA from protein
making intermediate ring of protein. As a cationic detergent, CTAB is readily soluble in water as
well as alcohol and can form complexes with both polysaccharide and residual protein.
7. Phenol:Chloroform:Isoamyl Alcohol
This is a method of liquid–liquid extraction. It separates mixtures of molecules based on
differential solubility of the individual molecules in two different immiscible liquids. Chloroform
mixed with phenol is more efficient at denaturing proteins than the only reagent. Chloroform
isoamyl alcohol is a type of detergent that binds to protein and lipids of cell membrane and
dissolves them. In this way, it disrupts the bonds that hold the cell membranes together. After
dissolving the cell membrane, chloroform isoamyl alcohol forms clumps of protein-lipid
complexes; thus, a precipitate is formed. The principle behind this precipitation is that, lipid–
protein complex are non-aqueous compounds and DNA is an aqueous compound. Thus, the upper
aqueous phase contains nucleic acid, middle phase contains lipids and the lower organic phase
contains proteins.
8. Isopropanol
DNA is highly insoluble in isopropanol, and hence, isopropanol dissolves in water to form a
solution that causes the DNA in the solution to aggregate and precipitate. Isopropanol is used as a
better alternative for ethanol due to its greater potential for DNA precipitation in lower
concentrations. Besides, it takes lesser time to evaporate.

PREPARATION OF REAGENTS AND BUFFERS

1. CTAB / NaCl solution (10% CTAB in 0.7 M NaCl)
Dissolve 4.1 g NaCl in 80 mL of distilled water and slowly add 10 g CTAB
(hexadecyltrimethyl ammonium bromide) by heating and stirring. Heat to 65⁰C to dissolve. Make
up the volume to 100 mL.
2. TE buffer (pH 8.0)
10 mM Tris.Cl, pH 8.0; 0.1 mM EDTA, pH 8.0. Store at room temperature.
3. 5 M NaCl
Dissolve 146.1 g of NaCl in 450 mL of distilled water and make up the volume to 500 mL.
4. 10% SDS
Dissolve 10 g of SDS in 80 mL of distilled water. Make up the volume to 100 mL.
5. Tris saturated phenol
Add equal volume of 1 M Tris pH 8.0 to liquid phenol and mix with glass rod. Allow to
settle. Separate the phenol layer and store for further use.
6. Chloroform : isoamyl alcohol (24:1)
Mix 1 mL of isoamyl alcohol with 24 mL of chloroform.
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M.Sc. Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

7. Phenol : chloroform : isoamyl alcohol (25:24:1)

Mix 25 mL of tris-saturated phenol with 24 mL of chloroform and 1 mL of isoamyl alcohol.
8. 70% ethanol
To 70 mL of ethanol, add 30 mL of sterile distilled water.
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EXPERIMENTAL PROCEDURE
1. Inoculate bacteria in 5 mL of LB broth by transferring a loop full from plate / slant culture.
2. Incubate the culture overnight at 37⁰C in a shaker incubator.
3. Transfer about 1.5 mL of liquid culture to microcentrifuge tubes and centrifuge at 8000
rpm for 5 min at 4⁰C or until a compact pellet forms.
4. Discard the supernatant. [Note: Remove as much of the supernatant as you can without
disturbing the cell pellet.]
5. Resuspend the pellet in 567/1134 µl TE buffer by repeated pipetting.
6. Add 30 µl of 10 % SDS and 3 µl of 20 mg/ml proteinase-K, mix thoroughly by inverting the
tubes, and incubate for 1 h at 37 °C in water bath.
7. Add 100 µl of 5 M NaCl and mix thoroughly. If NaCl concentration is < 0.5 M, then the
nucleic acid may also precipitate.
8. Add 80 µl of CTAB/NaCl solution and mix thoroughly by inverting the tubes.
9. Incubate the tubes in water bath at 65⁰C for 10 min.
10. Add 1 volume of 25:24:1 phenol/chloroform/isoamyl alcohol, extract thoroughly, and
centrifuge at 14,000 rpm for 5 min at 4⁰C.
11. Transfer the aqueous phase (supernatant) to a fresh tube.
12. Add 1 volume (0.7–0.8 ml) of 24:1 chloroform/isoamyl alcohol, mix thoroughly, and
centrifuge at 14,000 rpm for 4–5 min at 4⁰C.
13. Transfer the aqueous phase (supernatant) to a fresh tube.
14. To the supernatant, add 0.6 volume of isopropanol (i.e. 60 μL per 100 μL of sample) and
mix gently until a stringy white DNA precipitate forms and incubate at -20⁰C for 10 min.
15. Centrifuge at 12,000 rpm for 5 min at 4⁰C.
16. Discard the supernatant, and add 100 µl of 70 % ethanol to DNA pellet.
17. Centrifuge this mixture at 12,000 rpm for 2 min at 4⁰C
18. Discard the supernatant and air dry the DNA pellet by complete evaporation of ethanol (tilt
the tube a little bit on paper towel). Do not overdry.
19. Resuspend this dried DNA pellet in 50 µl of TE buffer to yield DNA solution. Typical yield is
5–20 µg DNA/ml of starting culture (108–109 cells/ml).
20. Check the purity of the DNA by agarose gel electrophoresis and UV-Visible
spectrophotometer and store at 4 °C in TE buffer till further use.
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OBSERVATION
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M.Sc. Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

The quality and quantity of the isolated DNA can be measured by agarose gel
electrophoresis and ultra-violet (UV)-visible spectrophotometer. For a 1-cm path length, the
optical density at 260 nm (OD260) equals 1.0 for the following solutions:
a. 50 µg/ml solution of double-stranded (ds) DNA
b. 33 µg/ml solution of single-stranded (ss)DNA
c. 20–30 µg/ml solution of oligonucleotide
d. 40 µg/ml solution of RNA
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Result Table
Sample DNA content OD260/280 Inference
Control
Sample I
Sample II
OD optical density
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PRECAUTIONS

1. Prepare wide bore pipette tips by cutting 2–3 mm from the ends and use them. This will
not allow DNA for mechanical disruption.
2. The incubation period with proteinase-K may be extended depending on the source of
DNA.
3. Repetition of phenol-chloroform extraction method should be performed to obtain a pure
DNA.
4. DNase-free plasticwares and reagents should be used during the entire procedure.
5. Phenol-chloroform is probably the most hazardous reagent used regularly in molecular
biology laboratories. Phenol is a very strong acid that causes severe burns. Chloroform is a
carcinogen. Handle these chemicals with care.
6. Wear gloves and goggles while isolating genomic DNA.
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TROUBLESHOOTINGS
Problem Possible cause Possible solutions
RNA contamination  If the bacterial density is too high,  Grow the bacterial cells ≤
i.e. more than 1 × 109 cells/ml, the 109 cells/ml
chances of RNA contamination
becomes more  Add RNase (400 µg/ml) to
 RNase is not added the isolated DNA sample
Protein contamination If the bacterial density is too high, i.e. Grow the bacterial cells ≤ 109
more than 1 × 109 cells/ml, the cells/ml

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M.Sc. Biotechnology, Date:
Analytical Techniques in Biotechnology Lab
Exp No:

chances of protein contamination Repeat the phenol:

becomes more chloroform: isoamyl alcohol
extraction step. Incubate the
mixture for 10 min at − 20 °C.
Centrifuge, discard
supernatant and add
500 µl 70 % ethanol
DNA concentration is Culture volume is too less Grow the bacterial culture
too less upto 109 cells/ml or collect
more pellet by repeated
centrifugation
Insoluble pellet after Error in methodology and the duration Extended drying under strong
DNA precipitation of drying the pellet vacuum may cause an
overdrying of the DNA. As an
acid, DNA is probably better
soluble in slightly alkaline
solutions such as TE or 10 mM
Tris buffer with a pH of 8.0
Degraded DNA Is the bacterial strain known as Do not let the bacterial
being “problematic”? culture grow for more than 16
h
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Reference:
1. Microbial Biotechnology- A Laboratory Manual for Bacterial Systems, by Surajit Das and
Hirak Ranjan Dash, Springer; 2015 edition.
2. Sambrook, J. and Russell, D. (2001) Molecular Cloning — A Laboratory Manual, 3rd Ed.,
Cold Spring Harbor Laboratory Press, New York.
3. Arora Dilip Kumar, Das Surajit, Sukumar Mesapogu (Eds). Analyzing Microbes, Manual of
Molecular Biology Techniques. Springer Protocols Handbooks, Springer; 2013 edition
(February 2, 2013)
4. http://wikieducator.org/ANDC_DU/Biology_Protocols/DNA_Spooling
5. Elisabeth Chachaty, Patrick Saulnier. Isolating Chromosomal DNA from Bacteria. The
Nucleic Acid Protocols Handbook, Ralph Rapley (Editors), Humana Press, 2000.

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