Exp No: 1 Date- 17/12/21

Isolation of Genomic DNA

AIM- To isolate genomic DNA from E. coli

THEORY- Genomic DNA is the chromosomal DNA present in the organism, inside nucleus in
Eukaryotes and in the nucleoid region in the prokaryotes. It contains the entire genetic information
of the cell.

Isolation of genomic DNA is done to separate the DNA of an organism away from proteins, RNA
and other cellular material.

Isolated Genomic DNA can be used for purposes like:

• Library construction
• Gene therapy
• Diagnostic of diseases
• SNP analysis

Owing to its applications it is necessary to isolate genomic DNA and many methods have been
developed to achieve this. Some of the methods are:

• Enzymatic method
• Phenol-chloroform method
• Kit based method (DNeasy Blood & Tissue) (purelink)

PRINCIPLE- Phenol- chloroform is an effective method is effective when it comes to isolation

of genomic DNA. Initially the cell membranes must be disrupted in order to release the DNA in
the extraction buffer SDS (sodium dodecyl sulphate). DNA can be protected from endogenous
nucleases by chelating Mg2++ ions using EDTA. Proteinase enzyme is used to degrade the
proteins in the disrupted cell mixture. 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

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

REQUIREMENTS-

• LB broth
• E.Coli cells
• TE buffer
• 10% SDS
• Proteinase K
• Phenol – Chloroform Mixture
• 5M sodium acetate

Miscellaneous-

• Microfuge
• Eppendorf tube
• Micro pipettes

PROTOCOL-

• 1.5 ml overnight culture is taken and the cells are harvested by centrifugation for 1 minute
• Add TE buffer is added to the cell pellet and the cells are resuspended in the buffer.
• 100 µl of 10% SDS and 5 µl of Proteinase K are added to the cells.
• The above mixture is mixed well and incubated at 37º C for an hour in an incubator.
• 1 ml of phenol-chloroform mixture is added to the contents, mixed well by inverting and
incubated at room temperature for 5 minutes.
• The contents are centrifuged at 10,000 rpm for 10 minutes at 4º C.
• The process is repeated once again with phenol-chloroform mixture and the supernatant is
collected in a fresh tube.
• 100 µl of 5M sodium acetate is added to the contents and is mixed gently.
• 2 ml of isopropanol is added and mixed gently by inversion till white strands of DNA
precipitates out.
• The contents are centrifuged at 5,000 rpm for 10 minutes.

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 • The supernatant is removed and 1ml 70% ethanol is added.
• The above contents are centrifuged at 5,000 rpm for 10 minutes.
• After air drying for 5 minutes 200 µl of TE buffer or distilled water is added.
• 10 µl of DNA sample is taken and is diluted to 1 or 2 ml with distilled water.
• The concentration of DNA is determined using a spectrophotometer at 260/280 nm.
• The remaining samples are stored for further experiments.

REFERNCE-

Genomic DNA Isolation. (2013). Iscan Cilga. Retrieved 24 January 2021, from
https://www.researchgate.net/publication/279511503_Genomic_DNA_Isolation

SCHOOL OF BIOENGINEERING DEPARTMENT OF BIOTECHNOLOGY BT0210 -

MOLECULAR BIOLOGY LABORATORYMANUAL FOR B.TECH BIOTECHNOLOGY
SEMESTER -IV. (n.d.). [online] Available at:
https://webstor.srmist.edu.in/web_assets/srm_mainsite/files/files/BT0210%20-
%20MOLECULAR%20BIOLOGY%20LABORATORYMANUAL.pdf [Accessed 17 Dec.
2021].

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Exp No. 2 Date – 17/12/21

Isolation of Plasmid DNA

AIM- Isolation of plasmid DNA from E.Coli.

THEORY-

A plasmid is a small, circular, piece of ds DNA molecule that is distinct from a cell's chromosomal
DNA and replicates autonomously. They are mainly found in bacteria, but they can also be present
in archaea and multicellular organisms. Often, the genes carried in plasmids provide the host with
genetic advantages, such as antibiotic resistance. They have a wide range of lengths, from roughly
one thousand DNA base pairs to hundreds of thousands of base pairs. When a bacterium divides,
all of the plasmids contained within the cell are copied such that each daughter cell receives a copy
of each plasmid.

Types of plasmid: There are different types of plasmid based on different classifying properties.
(I) Based on conjugation

(i) Conjugative plasmids: These types of plasmids can transfer genetic material from the
host to the recipient via sexual conjugation.
(ii) Non-conjugative plasmid: These plasmids cannot transfer genetic material via sexual
conjugation.

(II) Based on properties:

(i) F plasmid: These are also known as ‘F’- plasmids that contain transfer genes that allows
the genes to be transferred between bacteria (from donor to recipient) through
conjugation.
(ii) Resistance plasmid: These plasmids contain genes that help the host to defend against
extrinsic factors like antibiotics.

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 (iii) Degradative plasmid: These plasmids contain genes that helps the host to digest
compounds that are not common in nature.
(iv) Virulence plasmid: These plasmids contain virulence factors that makes the host act
like a pathogen.

Plasmid DNA is extensively used by scientists in genetic engineering and recombinant DNA
technology. Thus, there is a need of isolation of plasmids, for the following applications:

• Plasmids are used in genetic engineering to amplify copies of genes.

• They can be used in gene therapy to transfer genes.
• Can be used to transport genetic material between cells using a vector.
• It can be used to bulk produce antibiotics by incorporating an expression vector for that
antibiotic in microbial cells.

PRINCIPLE-

The most commonly used method for isolation of plasmid is alkaline lysis method. It uses the
property of difference in structural arrangement of plasmid and chromosomal DNA in the cell.
Plasmid DNA is in covalently closed form. A kit based method is used. In the presence of high
salt, plasmid DNA in cleared lysate binds selectively to glass microfiber membrane. This DNA is
purified using a series of steps to remove the additional cell debris. Finally Elution is done by a
low salt buffer. This simple method eliminates the need for organic solvent extraction and alcohol
precipitation.

PROTOCOL-

• Inoculate E.Coli in LB broth and incubate overnight at 37 degrees.

• Take 1.5 ml culture in an Eppendorf tube and centrifuge at 10000 rpm for 1 min at 4
degrees.
• Discard the supernatant and add 180 µl buffer and mix for 30 min at 37 degrees
• Take 20µl RNase and incubate at RT for 2min
• Add 20 µl Proteinase K
• Add 220 µl Binding buffer and store at 55 degrees for 30 min
• Add 220 µl of ethanol

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 • Transfer the mixture to the column of the kit.
• Centrifuge the column at 10000g for 30 sec
• Add 500 µl wash buffer 1 and centrifuge at RT for 1 min.
• Add 500 µl of wash buffer 2 and centrifuge at 10000 rpm for 30 sec
• Dry spin the column at 14000 rpm for 3 min
• Discard supernatant and transfer the column to new tubes and add 50 µl elution buffer and
incubate at RT for 1 min
• Centrifuge the tubes at 14000 rpm for 2min
• Perform electrophoresis with 0.8% agarose with 1XTAE bugger at 100v for 1hr
• Add 250 µl S1, 250 µl S2 and incubate at RT for 5 min then add 350 µl of S2
• Centrifuge at 14000 rpm for 10min at 4 degrees
• Transfer supernatant to the column and centrifuge for 1 min at 12000rpm at 4 degrees
• Add 500 µl of AW buffer wash and 700 µl PW buffer wash and spin for 2 min
• Add 50 µl elution buffer incubate for 1 min at RT and spin at 14000rpm for 1min at 4
degrees.

REFERENCE-

Genetic Education (2021). Retrieved 27 January 2021, from

https://geneticeducation.co.in/plasmid-dna-structure-function-isolation-and-applications/

7.4B: Types of Plasmids and Their Biological Significance. (2021). Retrieved 26 January 2021,

From
https://bio.libretexts.org/Bookshelves/Microbiology/Book:_Microbiology_(Boundless)/7:_Micro
bial_Genetics/7.04:_Plasmids/7.4B:_Types_of_Plasmids_and_Their_Biological_Significance

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