Aim: To isolate the genomic DNA from E .coli DH5α cells.

Requirements:
Biological: Overnight grown culture of E.coli (DH5α)
Chemicals
Luria-Bertani Broth (LB) medium, GTE mix, 10mM NaCl, 1%SDS, Isopropanol,
Ethidium bromide, TBE buffer (stock: 10X; working 0.5X), Agarose, Bromophenol
blue Loading dye, Ethidium bromide
Glassware and plasticware
Conical flask, Eppendorf, Micro tips, Pipettes
Instruments/ equipment
Orbital shaker, pH meter, Weighing balance, Autoclave, Centrifuge, Micropipettes,
Microwave/Hotplate, horizontal gel apparatus with power supply, UV
transilluminator,

Chemical Preparation
1. LB medium, 100 ml, pH 7.5
Tryptone1.0g, Yeast extract0.5g, Nacl0.5g, Glucose0.1g
Dissolve the components in 75ml of distilled water, adjust the pH to 7.6 and finally
make the volume to 100ml. Sterilize by autoclaving.
2. GTE Mix
Components Working Stock Volume (10ml)
Glucose 50mM 1M* 0.5ml
Tris-HCl pH 8 50mM 1M* 0.5ml
EDTA pH 8 10mM 0.5M* 0.2ml
Distilled water 8.8ml
*1M Glucose
Mol wt. 180g, 18g weighed and dissolved in 80ml distilled water. Volume raised to
100ml and autoclaved.
* 1M Tis-HCl buffer, pH 8.0
Mol wt. 121.1g, 12.11g Tris base weighed and dissolved in 60ml distilled water. pH is
adjusted by adding conc. HCl. Solution is allowed to cool before making final pH
adjustment. Volume raised to 100ml, filtered and autoclaved.
* 0.5M EDTA, pH 8
Mol wt. 372.2 g, 18.61 weighed and dissolved in 80ml distilled water. pH is adjusted
to 8.0 by adding NaOH. Volume raised to 100ml, filtered and autoclaved.
3. 10% SDS
Dissolve 10g in 100ml distilled water. Do not autoclave.
4. 1M NaCl:
Molecular weight: 58.44 g. Dissolve 5.84g in 100ml distilled water, filter sterilize.
Prepare 10mM NaCl from the stock solution by diluting in water.
5. Loading dye contains bromophenol blue, sucrose, EDTA pH8
BPB-0.25g, Sucrose-70g, EDTA-20ml. Dissolve required amount of sucrose in 50ml.
Add BPB and EDTA. Raise volume to 100ml.
6. TBE Buffer, pH 8.0 (10X) 1M Tris+1M Borate+0.02M EDTA
Tris (121.1g), Borate (61.8g), EDTA disodium salt (7.4g) for 1000ml Buffer
1X (500ml): Tris 6.05g, Borate 3.09g, EDTA disodium salt 0.37g
Principle:
The isolation of DNA is one of the most commonly used procedures in many areas of
bacterial genetics, molecular biology and biochemistry. 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. Bacterial strain used here is Escherichia
coli DH5α (gram-negative), with a genome size of 4x106 bp. DH5α cells are
engineered E. coli cells, developed by Douglas Hanahan as a cloning strain with high
transformation efficiency. The cells are defined by three mutations recA1 (disable the
activity of the recombinases and inactivate homologous recombination for stable
insert), endA1 (inactivates an intracellular endonuclease to prevent it from degrading
the future inserted plasmid) that help plasmid insertion and lacZM15 which enable
blue white screening.

1. Growing and harvesting a bacterial culture

The culture medium must provide a balanced mixture of the essential nutrients at
concentrations that will allow the bacteria to grow and divide efficiently.
Luria-Bertani (LB) is a complex or undefined medium, meaning that the precise
identity and quantity of its components are not known. This is because two of the
ingredients, tryptone and yeast extract, are complicated mixtures of unknown
chemical compounds. Tryptone supplies amino acids and small peptides, while yeast
extract (a dried preparation of partially digested yeast cells) provides the nitrogen
requirements, along with sugars and inorganic and organic nutrients.
Complex media such as LB need no further supplementation and support the growth
of a wide range of bacterial species. In LB medium at 37°C, aerated by shaking at
150–250 rpm on a rotary platform, E. coli cells divide once every 20 min or so until
the culture reaches a maximum density of about 2–3 x109 cells/ml.

In order to prepare a cell extract, the bacteria must be obtained in as small a volume
as possible. Harvesting is therefore performed by spinning the culture in a centrifuge.

2. Homogenization or disruption of cells

The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid
cell wall. These barriers have to be disrupted to release the cell components.
Techniques for breaking open bacterial cells can be divided into physical methods, in
which the cells are disrupted by mechanical forces such as sonication, tissue
homogenization and chemical methods, where cell lysis is brought about by
exposure to chemical agents that affect the integrity of the cell barriers. Chemical
methods are most commonly used with bacterial cells when the object is DNA
preparation.

Suspension of bacterial pellet in resuspension buffer, which contains Glucose, EDTA

and Tris.HCl.
• Glucose is required to make the solution isotonic, so that the cells do not
burst.
• Ethylenediamine tetraacetate (EDTA) chelate the divalent cations
(primarily magnesium and calcium) which are released upon bacterial lysis.
Removal of these cations destabilizes the cell membrane. It also inhibits
DNases.
• Tris.HCl acts as a buffering agent.
 • We can also add RNase A which is a very stable enzyme and is active under
very stringent condition including high alkaline condition, presence of
detergent and chelating agent (EDTA). Addition of RNase A in resuspension
buffer helps to remove RNA from the plasmid preparation.
Having lysed the cells, the final step in preparation of a cell extract is removal of
insoluble cell debris. Components such as partially digested cell wall fractions can be
pelleted by centrifugation, leaving the cell extract as a reasonably clear supernatant.

SDS is a detergent that solubilizes the phospholipid and denatures protein components
of the cell membrane, leading to lysis and release of the cell contents. Phenol-
chloroform, proteolytic enzymes such as Proteinase K are also used for dissociation of
nucleoprotein complexes in many cases.

3. Recovery of DNA from clear lysate/ Spooling of DNA

In the presence of salt (monovalent cations such as Na+, NH4+ or Li+), and at a low
temperature, absolute ethanol/isopropanol efficiently precipitates polymeric nucleic
acids. DNA is negatively charged, so adding a salt masks the charges and allows
DNA to precipitate. If enough ethanol is added, the electrical attraction between
phosphate groups and any positive ions present in solution becomes strong enough to
form stable ionic bonds and DNA precipitates. Centrifugation at high speed results in
collection of DNA in a form of pellet.
Alternatively, Spooling is an effective method of colletion of large amount of DNA
from an extraction process. Since DNA is water soluble, but insoluble in alcohol, thus
precipitated DNA is present at the interface of water-alcohol as milky translucent
mass clumped together. This entagled mass allows DNA to be visible and can be
spooped out with help of glass rod/microtip.

4. Washing of DNA pellet: Precipitation of DNA contains salts, which need to be

removed from the DNA. For this purpose, 70% ethanol wash is given to the pellet,
and air-dry for about 10-15 minutes to allow the EtOH to evaporate.

5. Storage of DNA: Obtained plasmid pellet can be dissolved in double distilled

water or Tris-EDTA (pH 8.0).

Qualitative Analysis of DNA

Agarose gel electrophoresis is a powerful and widely used method that separates
molecules on the basis of electrical charge, size, and shape. Buffers in gel
electrophoresis are used to provide ions that carry a current and to maintain the pH at
a relatively constant value. TBE or Tris/Borate/EDTA, is a buffer solution containing
a mixture of Tris base, boric acid and EDTA. Tris is a strong base and borate is an
acid, combination of both maintains the pH nearly neutral range of 8 to 8.5. Under
this alkaline condition, DNA is protected and can separate properly.
The agarose gel contains molecule sized pores, acting like molecular sieves. The
pores in the gel control the speed that molecules can move. DNA molecules possess a
negative charge in their backbone structure due to the presence of PO4- groups thus
this principle is exploited for its separation. Smaller molecules move through the
pores more easily than larger ones. Conditions of charge, size, and shape interact with
one another depending on the structure and composition of the molecules, buffer
conditions, gel thickness, and voltage. Agarose gels are made with between 0.7%
(provides good resolution of large 5–10 kb DNA fragments) and 2% (good resolution
for small 0.2–1 kb fragments).

The gel setup provides wells for loading DNA in to it. The loaded DNA molecules
move towards the positively charged electrode (anode) and get separated along the
length of the gel. Ethidium bromide (EtBr), a chromogen is added to the gel to
visualize the separated DNA under UV trans- illumination. EtBr intercalates between
the bases and glows when UV radiation is passed through the gel.

Purpose of gel loading buffer

The loading buffer gives colour and density to the sample to make it easy to load into
the wells. Also, the dyes are negatively charged in neutral buffers and thus move in
the same direction as the DNA during electrophoresis. This allows you to monitor the
progress of the gel. The gel loading dye possesses bromophenol blue. Density is
provided by glycerol or sucrose.

Procedure:
1. Take 1.5 ml of overnight grown culture of E.coli in microfuge tube.
2. Centrifuge at 10,000 rpm for 5 minutes to obtain pellet.
3. Discard the supernatant and add 200 µl of GTE mix. Break the pellet.
4. Incubate at room temperature for 5 minutes.
5. Add 400 µl of 1% SDS and invert mix.
6. Add 60 µl of 10mM NaCl and 700 µl of chilled isopropanol.
7. Invert mix. DNA threads are visible.
Option I
8. Centrifuge at 10,000 rpm for 20 minutes.
9. Decant the alcohol and air dry.
10. Dissolve in 200 µl of distilled water (MQ) and incubate at 45°C for 5-10mins.
11. Centrifuge at 10,000 rpm for 5 minutes.
12. Take the supernatant in a fresh microfuge tube and subject 20 µl of sample to
gel electrophoresis after adding 2µl BPB loading dye.
Option II
8. If sufficient DNA is visible, spool out with help of microtip and then airdry it
for 30 minutes at 45°C.
9. Add 50µl of MQ water for dissolution of DNA.
10. Incubate at 45°C for10mins and give a quick spin for few seconds to settle the
contents at bottom.
11. Take 10µl of supernatant and add 2µl of BPB loading dye and load the
samples for gel electrophoresis.

Preparation of Agarose Gel Electrophoresis

• Weigh 0.8 g agarose and dissolve it in 100 mL of 1x TAE Buffer (0.8%).
• Heat the solution over a hot plate to boiling constituency marked with a clear
solution
• Leave the solution to cool and add 5µl of EtBr solution mix it well by gentle
swirling.
• Pour it in the gel tray-comb set up. Also be sure the gel plates have been taped
securely and contain the well combs prior to pouring
• Allow the solution to cool and harden to form gel.
Loading of Samples
• Carefully transfer the gel to the electrophoresis tank filled with 1x TAE buffer.
• Prepare your samples [8 ul of DNA sample (0.1 ug to 1ug) and 2 ul of 5x gel
loading dye]
• Remove the comb and load the samples into the well.
• Connect appropriate electrodes to the power pack and run it at 50- 100volts for
20min.
• Monitor the progress of the gel with reference to tracking dye (Bromophenol blue).
Stop the run when the marker has run 3/4 th of the gel.
Examining the gel
• Place the gel on the UV-transilluminator and check for orange colored bands in the
gel.

Result:
A single band of genomic DNA was observed near the well in the agarose gel.
Paste the picture with proper labelling

Below figure Legend-Genomic DNA preparation from E.coli DH5α as visualized on

1% agarose gel with help of UV transilluminator

Discussion:

Nucleic acids are the most polar of the biopolymers and are therefore soluble in polar
solvents and precipitated by nonpolar solvents. The extracted DNA was dissolved in
distilled water. The quality of DNA was judged by the electrophoresis. On
electrophoresis one band was observed near the well, which clearly indicate that the
band is of high molecular weight and thus it’s genomic DNA. Since there only one
band is seen on the gel, so no shearing of DNA has taken place.
If a smear is seen, it is because of DNA shearing due to handling during preparation.
If RNAses is not used, RNA smear is also seen at the end of gel. If plasmid is also
present in bacterial cell, we will observe it as faster migrating bands, away from well.
Application of Genomic DNA
• Preparation of genomic libraries
• Use in PCR
• Gene cloning
• DNA sequencing, genome composition
• Performing various genetic studies such as DNA fingerprinting, gene therapy
• Studying DNA structure and chemistry
• Examining DNA-protein interactions
• Carrying out DNA hybridizations

Precautions:
1. Use thoroughly clean and sterile glassware and plasticware.
2. Use caution when operating centrifuge.
3. Wear gloves during the addition of EtBr and while handling the casted gel
(EtBr is a potent carcinogen).
4. Handling the gel should be careful as the gel may break due to improper
handling.
5. While performing the UV-trans illumination for visualising the bands, avoid
direct contact and exposure to eyes.