Biotechnology Engineering

1. ISOLATION AND
PURIFICATION OF TOTAL
CELLULAR DNA
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2. Why do we need DNA for?
• Detect/enumerate/ clone genes
• Detect/enumerate species
• Detect/sequence specific DNA regions
• To make recombinant DNA
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3. 3
For doing any recombinant DNA technology work,
one needs to isolate DNA or RNA from the cells of
organisms.
Total cellular DNA is isolated by lysing the cells with
a suitable physical or chemical method. The
contaminating biomolecules are removed by
extraction with organic solvents. Finally, the DNA is
precipitated with ethanol or isopropanol.

4. 4
Plasmid DNA can be separated from the chromosomal DNA by cesium
chloride-ethidium bromide density gradient centrifugation method.
Alternatively, alkali lysis method of H. C. Birnboim and J. Doly given in 1979
can be used. In this method, the cell lysis is carried out in presence of sodium
hydroxide at pH 12.5.
At this pH, the hydrogen bonds of both chromosomal DNA and plasmid DNA
are broken and single stranded DNA is obtained. Subsequently, pH of the
solution is lowered to 7.0.
At this pH, the plasmid DNA renatures whereas the linear single strands of
chromosomal DNA fail to find their complementary strands and get entangled
with the cell debris.
The cell debris containing the entangled chromosomal DNA is removed by
centrifugation. The plasmid DNA in the supernatant is precipitated with ethanol.
During the isolation of total or plasmid DNA, the problem of contaminating
DNases is tackled by sterile handling conditions and adding EDTA which
chelates the Mg2+ ions required for the activities of DNases.

5. Isolation and purification of total DNA from
bacterial cells
GROWING AND HARVESTING OF BACTERIAL CULTURE
BACTERIAL CELL LYSIS AND REMOVAL OF CELL DEBRIS
REMOVAL OF CONTAMINATING BIOMOLECULES FROM CELL EXTRACT
REMOVAL OF RNA CONTAMINATION
PRECIPITATION AND CONCENTRATION OF DNA
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6. 1) GROWTH OF BACTERIAL CULTURE
• Commonly used growth media – LB (Luria-Bertani), M9
• Growth conditions – For E. coli, grown in LB medium at
37ͦ C, aerated by shaking at 150-250 rpm.
2) HARVESTING OF BACTERIAL CELLS BY
CENTRIFUGATON
• After acquisition of appropriate cell density of bacterial
culture by monitoring Optical Density at 600nm
• OD = 1 corresponds to about 0.8X109 cells/ml
• Cells are harvested by spinning the culture at 3,000 to
8,000g for 5-10 minutes 6

7. LYSIS
PHYSICAL
CHEMICAL
VORTEXING, SONICATION
TREATMENT WITH
LYSOZYME,
EDTA, SDS
3) BACTERIAL CELL LYSIS AND REMOVAL OF
CELL DEBRIS
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8. DEPROTEINATION
PHENOL:CHLOROFORM
EXTRACTION
PROTEASE TREATMENT
PHENOL denatures proteins
CHLOROFORM facilitates
partitioning of aqueous and
organic materials
PRONASE / PROTEINASE K
Degrades proteins that are
easily removed by phenol
4) REMOVAL OF CONTAMINATING BIOMOLECULES
FROM CELL EXTRACT
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9. RNase TREATMENT
RNase A / RNase T1
5) REMOVAL OF RNA CONTAMINATION
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10. ETHANOL ISOPROPANOL
USING
BUTANOL
FOLLOWED
BY ETHANOL
6) PRECIPITATION AND CONCENTRATION OF
DNA
PRECIPITATION IS FOLLOWED BY DESALTING OF DNA PELLET
BY 70% ALCOHOL, RECENTRIFUGATION, AIR-DRYING AND
RESUSPENSION IN APPROPRIATE BUFFER OR WATER
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11. 1. CELL LYSIS
2. CENTRIFUGE TO REMOVE CELL DEBRIS
3. PHENOL:CHLOROFORM EXTRACTION 4. ETHANOL PRECIPITATION
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12. 2 ways to concentrate the genomic DNA
Spooling Ethanol precipitation 12

13. Choosing an Isolation Method
Important factors are:
– Processing speed
– Ease of use
– Yield of DNA or RNA
– Quality of DNA and RNA prepared
– Shelf life/storage conditions
– Cost of preparation
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