Aim: Isolation of genomic DNA from Bacterial cell

Introduction: All genetic information essential to the life of the bacterium is contained in a single
molecule of circular, double-stranded deoxyribonucleic acid (DNA), closed by covalent bonding.
This molecule is called a bacterial chromosome. Many bacteria also possess extrachromosomal
DNA, also circular and closed, called plasmid DNA because it is contained in the plasmids. These
carry gene information for many functions that are not essential for the cell under normal growth
conditions. The basic “standard” procedures for isolation of bacterial DNA are based on lysozyme
digestion of the cell wall, detergent lysis, disruption of protein-nucleic acid complexes and phenol:
chloroform extraction to remove proteins.

Figure: Structure of bacteria

Principle:

The isolation and purification of DNA from cells is one of the most common procedures in
contemporary molecular biology and embodies a transition from cell biology to the molecular
biology (from in vivo to in vitro). 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. The genomic DNA
isolation needs to separate total DNA from RNA, protein, lipid, etc. Initially the cell membranes
must be disrupted in order to release the DNA in the extraction buffer. SDS (sodium dodecyl
sulphate) is used to disrupt the cell membrane. Once cell is disrupted, the endogenous nucleases
tend to cause extensive hydrolysis. Nucleases apparently present on human fingertips are notorious
for causing spurious degradation of nucleic acids during purification. DNA can be protected from
endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary
cofactor for action of most of the nucleases. Nucleoprotein interactions are disrupted with SDS,
phenol or proteinase K. Proteinase enzyme is used to degrade the proteins in the disrupted cell
soup. Phenol and chloroform are used to denature and separate proteins from DNA. Chloroform
is also a protein denaturant, which stabilizes the rather unstable boundary between an aqueous
phase and pure phenol layer the denatured proteins form a layer at the interface between the

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aqueous and the organic phases which are removed by centrifugation. DNA released from
disrupted cells is precipitated by cold absolute ethanol or isopropanol.

Material required: Bacterial culture, Inoculating loop, Incubator, eppendorf tubes, sterilized
pipettes, sterilized tips, centrifuge, molecular grade water, autoclave, Phenol, chloroform, iso-amyl
alcohol (25:24:1 v/v), TE buffer (pH 8.0), 10 mM Tris-HCl, 1mM EDTA, 10% SDS (w/v), 5M
NaCl, Ethanol (70%), isopropanol, 70 % ethanol, hot air oven, tissue

Procedure:
Cellular lysis

1. Add 1.5 ml of an overnight culture to a 1.5 ml tube.

2. Centrifuge at 14,000 x g for 30 seconds. Remove the supernatant.

3. Add 600 μl of Lysis Solution to the cell pellet and pipette to resuspend and lyse the cells.

4. Incubate the samples at 60°C for 5-10 minutes. Cool at room temperature.

Protein purification

1. Add 300 μl of Phenol: Chloroform: Isoamyl alcohol solution.

3. Vortex vigorously for 20-30 seconds.

4. Centrifuge at 14,000 x g for 5 minutes.

DNA Precipitation

1. Transfer the supernatant containing the DNA to a 1.5 ml tube containing 600 μl of
isopropanol. Mix by inversion several times and incubate at -20℃ for 1 hr.

2. Centrifuge at 14,000 x g for 10 minutes.

3. Remove supernatant.
Washing

1. Add 600 μl of 70% ethanol and invert several times to wash the DNA pellet.
2. Centrifuge at 14,000 x g for 2 minutes. Carefully remove all the ethanol. Watch not lose the
DNA pellet.
3. Invert the tube and allow to dry on absorbent paper for 15 minutes.

Resuspension of DNA

1. Add 50-100 μl of TE buffer.

2. Resuspend by micropipette the white pellet. Incubation at 55°C with periodic vortex
stirring will aid in the dissolution of the DNA.
3. Store at 4°C

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 Figure: Steps involve in DNA extraction from bacterial cell.

Result: White thread like strands or white pellet of DNA can be seen.

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Aim: Isolation of genomic DNA from Plant cell

Introduction: Isolating Deoxyribonucleic Acid (DNA) from plant tissues can be challenging as
the biochemistry between divergent plant species can be extremely different. Unlike animal tissues
where the same tissue type from different species usually has similar characteristics, plant tissue
can have variable levels of metabolites and structural biomolecules. Polysaccharides and
polyphenols are two classes of plant biomolecules that vary significantly between species and are
problematic when isolating DNA. Contaminating polysaccharides and polyphenols can interfere
with manipulations of DNA following isolation. Plant DNA extraction and purification can be
divided into six steps: 1) tissue disruption/homogenization, 2) cell lysis in DNA extraction buffer,
3) separation of DNA from other cellular components, 4) DNA precipitation, 5) DNA washing,
and 6) DNA collection/resuspension for downstream processing.

Principle: DNA extraction involves lysis of cellular and nuclear membranes in order to extract
DNA from within. This is followed by DNA separation from impurities, proteins, and other
substances. General Stages of DNA Extraction are:

Cell dissolution: lysis of cell and the nucleus to extract DNA into the buffer.

Precipitation: removing the impurities and proteins from the sample.

Purification: This final stage is done to get a completely pure DNA sample ready to be used.
polysaccharides and polyphenols are problematic when isolating DNA from plant tissues.

CTAB buffers are effective at removing polysaccharides and polyphenols from plant DNA
preparations. CTAB (also called cyltrimethylammonium bromide) is a cationic detergent that
facilitates the separation of polysaccharides during purification while additives, such as
polyvinylpyrrolidone, aid in inactivating polyphenols. CTAB based extraction buffers are widely
used when purifying DNA from plant tissues. The hazard with traditional CTAB protocols is the
protein component of plant lysates is usually removed using phenol and chloroform.
Materials required: 2% CTAB buffer, 1% Polyvinylpyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl,
20 mM EDTA, Centrifuge, Isopropanol, 70% Ethanol, centrifuge tubes, TE Buffer (10 mM Tris,
pH 8, 1 mM EDTA), Phenol/Chloroform/Isoamyl Alcohol (25:24:1 ratio), Mortar and pestle

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 Figure: Steps involve in isolation of DNA from plant cells.

Procedure:

• Transfer the ground plant tissue to a polypropylene tube.

• For every 100 mg of homogenized tissue add 500 µl of CTAB Buffer. Mix and thoroughly
vortex.
• Place the tube in a 60°C water bath for 30 minutes.
• Centrifuge the homogenate for 5 minutes at 14,000 x g.
• Transfer supernatant to a new tube.
• Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).
• Vortex for 5 seconds then centrifuge the sample for 1 minute at 14,000 x g to separate the
phases.
• Transfer the aqueous upper phase to a new tube. Repeat this extraction until the upper phase
is clear.
• Transfer the upper aqueous phase to a new tube.
• Add 0.7 volume cold isopropanol and incubate at -20°C for 15 minutes to precipitate the
DNA.
• Centrifuge the sample at 14,000 x g for 10 minutes.

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 • Decant the supernatant without disturbing the pellet and subsequently wash with 500 µl ice
cold 70% ethanol.
• Decant the ethanol. Remove the residual ethanol by drying.
• Dry the pellet long enough to remove alcohol, but without completely drying the DNA.
• Dissolve the DNA pellet in 20 µl TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The pellet
may need to be warmed, in order to dissolve.

Result: The DNA appears as white precipitates of fine thread on the spool.

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Aim: Isolation of RNA from bacterial cell.

Introduction: Obtaining high-quality RNA is the first, and often the most critical, step in
performing many molecular techniques such as reverse transcription real-time PCR (RT-qPCR),
transcriptome analysis using next-generation sequencing, array analysis, digital PCR, northern
analysis, and cDNA library construction. To generate the most sensitive and biologically relevant
results, the RNA isolation procedure must include some important steps before, during, and after
the actual RNA purification. To isolate RNA from bacterial cells, a common method involves cell
lysis, protein and DNA removal, and RNA precipitation. This typically involves using a reagent
like TRIzol or a similar lysis solution, followed by chloroform and isopropanol for extraction and
precipitation. Washing the RNA pellet with ethanol and resuspending in a buffer like TE completes
the process.

Principle of RNA Isolation

Total RNA is isolated and separated from DNA and protein after extraction with a solution called
as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and
chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and
proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered
by precipitation with isopropanol.
Materials required: Bacterial culture, Trizol, chloroform, isopropanol solution, TAE buffer,

70% ethanol, centrifuge tube, centrifuge

Figure: Steps involve in RNA isolation.

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Procedure:

• Grow an overnight culture of bacteria in the appropriate media and at the appropriate
temperature.
• Centrifuge 1ml of culture, in a 1.5ml tube, at 14,000 x g for 2 minutes at room
temperature.
• Carefully remove the supernatant, leaving the pellet as dry as possible.
• To this add 160 μL of Trizol (1/5th of culture volume).
• The solution was mixed well by pipetting several times.
• To this add 32 μl of chloroform (1/5th volume of trizol).
• Incubate for 2 to 5 minutes and centrifuge at 12000 rpm for 15 minutes at 4° C
• Transfer the aqueous phase into a new tube and add equal volume of isopropanol. Mix
well. Incubate for 1 hr. at 4° C
• Centrifuge at 10000 rpm for 10 minutes at 4° C.
• Discard the supernatant and resuspend the pellet in 70% ethanol.
• Again centrifuge at 10000 rpm for 10 minutes at 4° C.
• Discard the supernatant.
• Air dry the pellet at 37° C for 10-15 minutes.
• Resuspend the pellet in 50 μL of TE buffer.
• Analyse the RNA sample quantitatively and qualitatively.
Results: A gel-like pellet on the side and bottom of the tube of RNA precipitate. On taking OD
at 260 nm and 280 nm, the A260/A280 ratio should be above 1.6.

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