! =>H=JHO =K= BH /AAJE? -CEAAHEC
`
`5
`
`4AIJHE?JE,ECAIJE=@EC=JEB,)
`
`Restriction enzyme digestion is performed by incubating double-stranded DNA molecules with
`an appropriate amount of restriction enzyme, in its respective buffer as recommended by the
`supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride
`concentration in the reaction varies for different enzymes, and a set of three-standard buffers
`containing three concentrations of sodium chloride are prepared and used when necessary.
`Typical digestions include a unit of enzyme per microgram of starting DNA, and one restriction
`enzyme unit is usually defined as the amount of enzyme needed to completely digest one
`microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions
`usually are incubated for 1–3 hours, to ensure complete digestion, at the optimal temperature for
`enzyme activity, typically at 37°C.
`DNA ligations are performed by incubating DNA fragments with appropriately digested
`cloning vector in the presence of ligation buffer containing ATP and T4 DNA ligase. For random
`shotgun cloning, sonicated or nebulized fragments, in which the fine mist created by forcing a
`DNA solution through a small hole in the nebulizer unit is collected. The size of the fragments
`obtained by nebulization is determined chiefly by the speed at which the DNA solution passes
`through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of
`the solution, and the temperature. Nebulization is easy, quick, and requires only small amounts
`of DNA (0.5–5 mg). The resulting DNA fragments are distributed over a narrow range of sizes
`(700–1330 bp). It requires ligation of DNA before nebulization and end-repair afterward, are
`ligated either to Sma I linearized, dephosphorylated double-stranded M13 replicative form or pUC
`vector by incubation at 4°C overnight. A practical range of concentrations is determined based
`on the amount of initial DNA and several different ligations, each with an amount of insert DNA
`within that range, are used to determine the appropriate insert to vector ratio for the ligation
`reaction. In addition, several control ligations are performed to test the efficiency of the blunt-
`ending process, the ligation reaction, and the quality of the vector. These usually include parallel
`ligations in the absence of insert DNA to determine the background clones arising from self-
`ligation of inefficiently phosphatased vector. Parallel ligations are also performed with a known
`blunt-ended insert or insert library, typically an Alu I digest of a cosmid, to ensure that the blunt-
`ended ligation reaction would yield sufficient insert containing clones, independent of the repair
`process.
`
`32
`
`00001
`
`EX1023
`
`

`

`Restriction Digestion and Ligation of DNA !!
`
`5.1 RESTRICTION DIGESTION OF DNA
`
`Protocol
`1. Prepare the restriction digestion reaction mixture by adding the following reagents in the
`order listed to a microcentrifuge tube:
`= 10 mL
`DNA (1 mg)
`2 mL
`=
`10X restriction enzyme assay buffer
`1 mL
`=
`Restriction enzyme (1–10 units)
`7 mL
`=
`Sterile ddH20
`Total restriction volume = 20 mmmmmL
`
`Note
`(i)
`
`If desired, more than one enzyme can be included in the digest if both enzymes are
`active in the same buffer and the same incubation temperature.
`(ii) The volume of the reaction depends on the amount and size of the DNA being
`digested. Larger DNAs should be digested in larger total volumes (between
`50–100 mL).
`(iii) The supplier’s catalogue should be referred to the chart of enzyme activity in a
`range of salt concentrations to choose the appropriate assay buffer (10X High, 10X
`Medium, or 10X Low salt buffers, or 10X Sma I buffer for Sma I digestions).
`2. Gently mix the restriction digestion reaction mixture by pipetting up and down and
`incubate the reaction at the appropriate temperature (typically at 37°C) for 1–3 hours.
`3. After incubation for 1–3 hours inactivate the enzyme(s) by heating at 65°C for
`10 minutes or by phenol extraction. Prior to use for subsequent dephosphorylation or
`ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis
`along with non-digested DNA and a size marker, for conformation of the digestion
`(Figure 5.1).
`
`5.2 PURIFICATION OF RESTRICTED DNA FRAGMENTS
`
`Protocol
`1. Dilute the restricted DNA to 500 mL with TE buffer.
`2. Extract once with equal volume of equilibrated phenol (add equal volume of phenol, mix
`and centrifuge at 10,000 rpm for 10 minutes at 4°C. Transfer the upper phase into
`another sterile tube).
`3. Add equal volume of chloroform: isoamyl alcohol (24:1) and extract as in previous step.
`4. Take the upper phase and add sodium acetate (pH 4.6) to a final concentration of 0.3 M.
`Mix well and add four volume of ice cold 100% ethanol.
`5. Mix well and incubate for 1 hour at –20°C and centrifuge at 10,000 rpm for 10 minutes
`at 10°C.
`6. Decant the supernatant and wash the DNA pellet with 70% ethanol.
`7. Air dry the final DNA pellet and resuspend it in 10 mL of sterile double distilled water
`for subsequent ligation reaction or 10 mL of TE buffer for storage.
`
`00002
`
`

`

`!" =>H=JHO =K= BH /AAJE? -CEAAHEC
`
`M: 1 mg f X 174/HaeIII Markers
`A: Undigested DNA;
`B: BpuAm1
`C: Sac 1
`
`M
`
`A
`
`B
`
`C
`
`Figure 5.1 Agarose gel electrophoresis of restriction fragments produced by cleavage of Ad2
`phage DNA.
`
`Buffer
`(i) 3 M Sodium acetate (pH 4.6)
`(Dissolve sodium acetate salt in less volume of distilled water and adjust the pH to 4.6
`with glacial acetic acid and finally make up the volume).
`
`5.3 DNA LIGATION
`
`Protocol
`1. Combine the following reagents in a microcentrifuge tube and incubate overnight at
`12–16°C:
`
`Digested insert DNA fragments
`Digested cloning vector
`(with same restriction enzymes)
`10X ligation buffer
`T4 DNA ligase
`Sterile ddH2O
`
`Total volume
`
`=
`=
`
`4 mL (50 ng/mL)
`2 mL (10 ng/mL)
`
`1 mL
`=
`1 mL (10 U/mL)
`=
`2 mL
`=
`= 10 mmmmmL
`
`2.
`
`Include control ligation reactions with no insert DNA and with a known blunt-ended
`insert (such as Alu I digested cosmid) as controls.
`
`00003
`
`

`

`Restriction Digestion and Ligation of DNA !#
`
`3. Transform the ligated DNA in to E. coli either by competent cell transformation or by
`electroporation.
`4. Score the efficiency of ligation.
`
`REFERENCES
`
`Ausubel, F.M., et al. (1994–2000), Current Protocols in Molecular Biology, vol. 1, John Wiley
`& Sons, Inc., Brooklyn, New York.
`Sambrook, J. and D.W. Russel (2001), Molecular Cloning: A Laboratory Manual, 3rd ed., Cold
`Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press.
`
`00004
`
`

`

`Laboratory Manual
`for
`
`Genetic
`Engineering
`
`S. John Vennison
`S. John Vennison
`
`00005
`
`

`

`Laboratory Manual
`for
`Genetic Engineering
`
`S. John Vennison
`Lecturer
`Department of Biotechnology
`Anna University
`Tiruchirappalli
`
`New Delhi-110001
`2009
`
`00006
`
`

`

`LABORATORY MANUAL FOR GENETIC ENGINEERING
`S. John Vennison
`
`© 2009 by PHI Learning Private Limited, New Delhi. All rights reserved. No part of this book may
`be reproduced in any form, by mimeograph or any other means, without permission in writing from
`the publisher.
`
`ISBN-978-81-203-3814-2
`
`The export rights of this book are vested solely with the publisher.
`
`Published by Asoke K. Ghosh, PHI Learning Private Limited, M-97, Connaught Circus,
`New Delhi-110001 and Printed by Glorious Printer, Delhi-110092.
`
`00007
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`

`

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`
`
`Contents
`
`5
`
`Preface .......................................................................................................................................................................... vii
`1. Isolation of Genomic DNA from GRAM Negative, GRAM Positive
`Bacteria, Blood and Mammalian Tissue ....................................................................... 1-6
`1
`1.1 Total DNA Extraction from Agrobacterium Tumefaciens and E. coli
`2
`1.2 Total Cell DNA Isolation from Bacillus Thuringiensis
`4
`1.3 Genomic DNA Isolation from Blood
`1.4 A Rapid Procedure for Isolation of DNA Cultured Mammalian Cells
`References
`6
`2. Isolation of Plasmid DNA from GRAM Negative and
`GRAM Positive Bacteria ............................................................................................... 7-21
`7
`2.1 Growth of Bacteria
`2.2 Harvesting and Lysing of Bacteria
`2.3 Purification of the Plasmid DNA
`9
`2.3.1 Cloning in Plasmid Vectors
`9
`2.3.2 Cloning DNA Fragments with Protruding Ends
`9
`2.4 Modified Alkaline Lysis Protocol for Both E. coli and Bacillus sp.
`2.5 High Molecular Weight Plasmid Preparation from GRAM Negative Bacteria
`(Including Agrobacterium Tumefaciens)
`11
`12
`2.6 Plasmid Isolation from E. coli (Mini Preparation)
`2.7 Large Scale Plasmid Preparation for E. coli (Alkaline Lysis)
`14
`2.7.1 Plasmid Amplification
`14
`2.7.2 Harvesting
`14
`2.7.3 Lysis with Alkali
`14
`2.8 Rapid Boiling Method of Isolation of Bacterial Plasmid
`15
`2.9 Isolation of Plasmid DNA from Bacillus Thuringiensis
`2.10 Purification of High Molecular Weight Plasmids from Bacillus Thuringiensis
`2.11 Isolation of Plasmid DNA from Bacillus Subtilis and Bacillus Megaterium
`2.12 Purification of Genomic and Plasmid DNA through Phenol
`17
`Chloroform Treatment
`
`8
`8
`
`iii
`
`13
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`16
`17
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`00008
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`

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`iv Contents
`
`
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`
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`30
`
`33
`
`39
`
`2.13 Ultra Purification of Plasmid DNA through Cesium Chloride
`Ethidium Bromide Gradient 18
`References
`20
`3. Isolation of RNA from Bacteria and Cultured Mammalian Cells ........................ 22-25
`23
`3.1 Isolation of Bacterial RNA
`3.2 Isolation of mRNA from Cultured Mammalian Cells
`References
`25
`4. Estimation of Nucleic Acids ........................................................................................ 26-31
`26
`4.1 Estimation of DNA
`4.1.1 UV Quantitation of DNA by UV Absorbance Spectrophotometry
`27
`4.1.2 TD-20/20 Luminometer Method for DNA Quantitation
`29
`4.1.3 Diphenylamine Method
`30
`4.2 Estimation of RNA
`4.2.1 Orcinol Method
`References
`31
`5. Restriction Digestion and Ligation of DNA ............................................................. 32-35
`33
`5.1 Restriction Digestion of DNA
`5.2 Purification of Restricted DNA Fragments
`34
`5.3 DNA Ligation
`References
`35
`6. Polymerase Chain Reaction and Randomly Amplified Polymorphic DNA ......... 36-40
`36
`6.1 Important Parameters in the PCR
`6.1.1 Tm of Primers
`37
`37
`6.1.2 Mg Concentration
`37
`6.1.3 Length of Expected Product
`38
`6.2 Polymerase Chain Reaction (PCR)
`6.3 Random Amplified Polymorphic DNA (RAPD)
`References
`40
`7. Electrophoresis of Nucleic Acids ................................................................................ 41-49
`41
`7.1 Agarose Gel Electrophoresis of DNA
`7.1.1 The Rate of Migration of DNA through Agarose Gels
`46
`7.2 Polyacrylamide Gel Electrophoresis of DNA
`7.3 Electrophoresis of RNA through Gels Containing Formaldehyde
`References
`49
`8. Slot Lysis Agarose Gel Electrophoresis ..................................................................... 50-54
`50
`8.1 Horizontal Slot Lysis Electrophoresis for E. coli
`8.2 Vertical Slot Lysis Electrophoresis for B. Thuringiensis
`51
`(Modified Eckhardt's Lysate Electrophoresis)
`References
`54
`
`24
`
`26
`
`42
`
`48
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`00009
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`
`
`Contents v
`
`56
`
`61
`
`62
`64
`
`9. Purification of DNA from Agarose and Polyacrylamide Gels ............................... 55-56
`55
`9.1 Isolation of DNA from Agarose Gels
`9.2 Isolation of DNA Fragments from Polyacrylamide Gels
`Reference
`56
`10. Transformation of GRAM Negative and GRAM Positive Bacteria
`with plasmid DNA........................................................................................................ 57-68
`57
`10.1 Competent Cell Transformation of Gram Negative Bacteria
`58
`10.2 E. coli Transformation by Calcium Chloride Method
`59
`10.3 E. coli Transformation by TSB Buffer Method
`60
`10.4 E. coli Transformation by Electroporation
`10.5 Simple Method of Plasmid Transformation of E. coli by Rapid Freezing
`61
`10.6 Protoplast Transformation of Bacillus sp. with Plasmid DNA
`10.7 Protoplast Transformation of Bacillus sphaericus with Plasmid DNA
`10.8 Competent Cell Transformation of Bacillus subtilis with Plasmid DNA
`66
`10.9 Transformation of Bacillus Thuringiensis by Electroporation
`References
`67
`11. Estimation of Proteins ................................................................................................. 69-71
`69
`11.1 Estimation of Protein by Bradford's Method
`70
`11.2 Estimation of Protein by Lowry's Method
`References
`71
`12. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Proteins ...... 72-78
`73
`12.1 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis of Proteins
`77
`12.2 Silver Staining of Protein Gels
`References
`78
`13. -Galactosidase Assay ................................................................................................. 79-81
`80
`13.1 Disruption of Selective Permeability
`81
`13.2 Enzyme Assay
`Reference
`81
`14. Transduction of Plasmid DNA using CP-51 and CP-54 Bacteriophages ............. 82-85
`82
`14.1 Transduction of Plasmid in Bacillus sp. with CP-51 and CP-54 Phage
`References
`85
`15. Bacterial Conjugation .................................................................................................. 86-89
`86
`15.1 Conjugal Transfer of DNA into Cyanobacteria
`15.2 Conjugal Plasmid Transfer in B. Thuringiensis
`87
`15.3 Introduction of Binary Plasmids into Agrobacterium by Triparental Mating
`References
`89
`16. Blotting Techniques ................................................................................................................ 90-99
`90
`16.1 Western Blotting
`16.2 Immunoblotting Assay
`
`88
`
`93
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`00010
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`vi Contents
`
`
`
`94
`
`94
`16.3 Southern Blotting
`16.3.1 Capillary Blotting or Passive Diffusion Blotting on Nitrocellulose
`96
`16.3.2 Southern Blotting using Semiphor Blotting Unit
`98
`16.3.3 Colony Blotting
`99
`16.4 Northern Blotting
`References
`99
`17. 32P Labelled Probe Preparation and Measurement of Radioactivity in
`Radio-Labelled Nucleic Acid .................................................................................. 100-102
`100
`17.1 32P Labelled Probe Preparation
`17.1.1 Nick Translation or Oligolabelling with a32P dCTP
`17.1.2 Random Primer Labelling of Probe using a32P dCTP
`17.1.3 Separation of Probe from Unincorporated Label by Gel-
`101
`Filtration through Sephadex G-50 Column
`17.2 Measurement of Radioactivity in Nucleic Acid
`101
`17.2.1 Absorption to DE-81 Filter
`17.2.2 Precipitation with Trichloroacetic Acid (TCA)
`Reference
`102
`18. Hybridization Techniques ............................................................................................. 103-106
`103
`18.1 Hybridization of Southern Filters
`103
`18.1.1 Prehybridization
`105
`18.2 Colony Hybridization
`References
`106
`Appendices ........................................................................................................................ 107-124
`109
`1. Stock Solutions and Working Concentrations of Antibiotics
`110
`2. Conversion of rpm to g
`3. Conversion of g to rpm
`111
`112
`4. Stock Solutions
`117
`5. DNA/Protein Conversions
`118
`6. Common Conversions of Oligonucleotides
`120
`7. Restriction Enzymes and their Cleavage Sites
`8. Estimation of Ends (3' or 5') Concentration
`122
`9. Recommended Gel Percentages for Separation of Linear DNA
`124
`10. Calculating Primer Quantity
`
`101
`
`102
`
`100
`101
`
`123
`
`00011
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