techniques in molecular biology and dna technology
TRANSCRIPT
PROJECT REPORT
ON
TECHNIQUES IN MOLECULAR
BIOLOGY AND DNA
TECHNOLOGY
PRESENTED BY
SHAQUIB SALIM
III YEAR BSC BIOTECHNOLOGY
REG NO 022UDO61
February 17, 2004
C E R T I F I C A T E
This is to certify that Mr. shaquib salim studying in III year B.Sc.
Biotechnology at CMR Institute of Management Studies, Bangalore, has
Successfully completed his ‘On Job Training’ titled Techniques in Molecular
Biology and DNA Technology in the Department of Molecular Biology at
Jain Institute of Vocational & Advanced Studies, Bangalore.
We wish him success in his future endeavour.
Date: 17.02.2004
Place: Bangalore
CERTIFICATEThis is to certify that the Project report entitled “
Techniques in Molecular Biology and DNA Technology ” prepared and submitted by Mr.Shaquib Salim (Reg No 022UDO61) in partial fulfillment for the award of Degree in Bachelor of Science (Biotechnology), is a record of bonafide work done by him during period of the study 2003-04 in CMR Institute of Management Studies.
This report has not been previously utilized as the basis for the award of any Degree/Diploma/Fellowship or any other similar title of any other University.
College Seal Principal.
[ Dr.A.MUKHOPADHYAY]
C.M.R. INSTITUTE OF MANAGEMENT STUDIES
We offer our thanks and gratitude to Mr. D.P. Prakash, for allowing us to work in the laboratory under his strict supervision and guidance.We take this opportunity with immense pleasure to express our sense of gratitude to Mr.Danis.G.Lalan for assisting us in the laboratory and providing every possible facility and guidance.
Our grateful thanks and sincere gratitude to Mrs. Shobha Murthy, who has provided us sufficient reference books and constant association during this project work.
With immense pleasure we extend our thanks and gratitude to HOD and the Dean of Bio-Science department Dr. Mukhopadhay for his guidance and supervision.
SUBMITTED BY:
SHAQUIB SALIM
DATE:24.02.2004
III Bsc Biotechnology
CMRIMS
OBJECTIVE
To study Molecular biotechnology using various rDNA
techniques such as Isolation of DNA, Electrophoresis, Gel
elution, Ligation, Genomic DNA in plant, etc.
Learning these techniques would help a Molecular biotechnology
student to explore the avenue of which is very integral part of
any human science
CONTENTS:
Sl
no TOPICS Page No
1 INTRODUCTION TO MOLECULAR BIOLOGY 01-07
2 EVENTS AND DISCOVERIES 08-11 REVIEW OF LITERATURE
3 A) PLASMID DNA 13-19 B) RESTRICTION ENDONUCLEASE 20-23
C) LIGATION 24-25D)GEL ELECTROPHORESIS 26-29
4 INSTRUMENTATION 30-49
MATERIAL, METHOD, OBSERVATION, RESULT AND DISCUSSION
A)ISOLATION OF PLASMID DNA FROM E.coli 51-58B) EXTRACTION OF GENOMIC DNA FROM PLANT
MATERIAL 59-62C) RESTRICTION DIGESTION 63-68D)LIGATION 69-75E) AGAROSE GEL ELECTROPHORESIS 76-79F) GEL ELUTION 80-84G)ISOLATION OF TOTAL RNA 85-88
6 SUMMARY 89-91
7 GLOSSARY 92-93
8 APPENDIX 94-96
9 REFERENCE 97-98
The term molecular biology was first used in 1945 by William Astbury who was referring to the study of the chemical and physical structure of biological macromolecules. By that time, biochemists had discovered many fundamental intracellular chemical reactions. The importance of specific reactions and of protein structure in defining the numerous properties of cells was also appreciated. However, the development of molecular biology had to await the understanding that the most advantageous approaches would be made by studying simple systems such as bacteria and bacteriophages, which yield information about the basic biological uniformity of life processes was an important factor in rapid growth of molecular biology. That is, it was believed that the fundamental biological principles that govern the activity of simple organisms, such as bacteria and viruses, must apply to more complex cells; only the details should vary. This faith has been amply justified by experimental results.
The roots of molecular biology were established in 1953 when an Englishman, Francis Crick and a young American, James Watson working at medical research council unit, Cavendish laboratory, Cambridge, proposed a double helical model for the structure of DNA molecule which was well known as chemical bearer of genetic informations of certain microorganisms due to pioneer discoveries made by Grifith(1928), Avery, McLeod and Mc Carthy (1944) and Harshey and Chase(1952). This discovery was followed by a thorough search of occurrence of DNA as genetic material in other microorganisms, plants and animals and also by investigation of the molecular and atomic nature of different reactions of living cells. From all these studies has emerged the realization that the basic chemical organization and the metabolic processes of all living things as remarkably similar despite their morphological diversity and that the physical and chemical principles
governing living systems are similar to those governing non-living systems.
The present understanding of molecular biology is that in most organisms the phenotype or the body structure and function ultimately depend for their determination on the structural and functional proteins or polypeptides. The synthesis of polypeptides is specified, directed and accepted chemical bearer of genetic information of most living organisms except certain viruses in which this function is carried by RNA, another nucleic acid. The genetic information for polypeptide synthesis are initially dictated by the deposition of nitrogen bases in DNA molecule and are copied down by the processes of transcription. During transcription stage copies of an individual gene or genes are synthesized. These copies are molecules of RNA that include such familiar classes as ribosomal RNA, messenger RNA and transfer RNA. The biochemical interplay of these RNA classes which leads to the synthesis of a polypeptide chain, is called translation, meaning, literally, that the genetic message encoded in a messenger RNA molecule is translated into the linear sequence of amino acids in a polypeptide. The polypeptide in its turn determines the phenotype of the organism.
HISTORICAL BACKGROUND
The molecular biology is a very young biological discipline and has a very short history. Certain notable accomplishments of molecular biologists can be summarized as follows:
1928: F. Griffith demonstrated that the bacteriophages are composed of DNA and protein.
1944: O.T.Avery, C.M.Macleod and M.Maccarthy recognized the nature of transforming principles of pneumococcus bacteria. The fact suggested that it is DNA and not protein, which is the hereditary chemical.
1950: E. Chargaff demonstrated that in DNA the numbers of adenine and thymine groups are always equal and so are the numbers of guanine and cytosine groups.
1953: J.D.Watson and F.H.C.Crick proposed a model for DNA comprising of two helically intertwined chains tied together by hydrogen bonds between the purines and the pyrimidines.
1956: A. Gierer and G.Schramm demonstrated that RNA is the genetic material of tobacco mosaic virus.
1958: J. Laderberg got the Nobel Prize for the discovery of bacterial recombination.
1961: F.H.C. Crick and his colleagues showed that the genetic material language is made up of three letter words, which are called as triplet codons.
Jacob and Monod put forward the operon concept.
1962: J. Watson and F. Crick; M. Wilkens got Nobel Prize for the discovery of molecular nature of DNA.
1965: F. Jacob, A. Lwoff and J. Monod received Nobel Prize for the discovery of protein synthesis mechanism in virus.
1968: R.W.Holley, H.G.Khorana and M.W.Nirenberg got Nobel Prize for deciphering the genetic code.
MATERIALS AND METHODS IN MOLECULAR BIOLOGY
Different molecular biologists have made an intensive use of a variety of the microorganisms such as bacteriophages and other viruses, E.Coli and other bacteria; unicellular green algae, yeast, neurospora, and other fungi; protozoans, etc, in their investigation of varied nature. However, working on larval salivary gland chromosomes of dipteran insect and lampbrush chromosomes of oocytes of different amphibians and other higher animals has made many significant discoveries of molecular biology. To understand different biochemical events of prokaryotes and eukaryotic cells at molecular level, a wide array of bio-physio-chemical techniques such as electron microscopy, ultra centrifugation, calorimetry, spectrophotometry, chromatography, isotopic tracers, X-ray crystallography, electrophoresis, etc. are used in molecular biology.
BASIC REQUIREMENTS TO BE MET BY GENETIC MATERIAL
According to the molecular biologists any molecule must meet certain requirements if it is to be qualified as the substance that transmits genetic information from one generation to the next. These requirements extended directly from what is known about the continuity of species and the processes of evolutionary change.
1. The genetic material must contain biological useful information that is maintained in a stable form.
2. The genetic information must be able to express itself so that other biological molecules, and ultimately cells and organisms, will be produced and maintained. Implicit in these requirements is that
some mechanisms be available for decoding, or translating the information contained in the genetic material into its productive form. A narrow but important distinction is thus made between molecules that can generate only its own kind and a molecule that can also generate new kinds of molecules. For example, a salt crystal can seed a salt solution so that new salt crystals are formed, but this is the extent of its influence over its surroundings.
3. The genetic information must be reproduced and transmitted faithfully from cell to cell or from generation to generation.
4. Genetic material must be capable of variations. Two sources of change have been recognized in present day genetic systems: mutation and recombination. A mutation changes the nature of information transmitted from parent to offspring and thus it represents a relatively drastic way of bringing about variation. Recombination is a more moderate way of producing variations. It occurs during the course of some sort of sexual process, and it involves the precise shuffling of parental genetic information so that new combinations of genes are produced. These are then inherited by the offspring.
5. With these four requirements in mind we can study most of the aspects that is related to genes its structures and its transformations.
1944 – Ostwald Avery discovers genetic information is carried by nucleic acids of cells.
1952 – Joshua Lederberg introduced the word Plasmid and defined it as an extra chromosomal element.
1953 – Watson and Crick proposed the double stranded structure model of DNA
- DNA was universally accepted as genetic material.
1960 – Warner Arber postulated the presence of restriction enzyme.
1961 – Successful attempt by Nirenberg and Mathaei to break the genetic code.
1966 – Establishment of the complete genetic code.
1967 – Enzyme DNA LIGASE was isolated
1968 – Shapiro and Beckwith isolated the first gene.
1970 – Khorana and his colleagues succeeded in synthesizing gene in the test tube by assembling its constituents base pair and foundation for recombinant DNA technology.
-First restriction enzyme was isolated.
-Sgaramella et al. reported that T4 ligase enzyme can joint (blunt end) fully base paired DNA duplex.
1972 – Paul Berg creates the first recombinant DNA molecules.
1979 – Birnboin and Doly described alkaline lysis method.
1983 – Kruger and Bickle found that bacteriophage commonly have antirestriction mechanism of one sort or the other.
1986 – the structure of enzyme – DNA complex has been determined by X-ray crystallography by McClarin et al.
1988 – Dower et al. found that Electroporation works well with most commonly used laboratory strain of E.Coli.
1990 – Robert identified about 150 type II restriction endonuclease and partially characterized them.
1991 – Wilson and Murray reviewed the biochemical evolutionary relationships of restriction modification systems.
1992 – Sutherland et al. found the cleavage required GTP.
1993 – Murray et al. found the evidences for selection for favoring divergence of restriction specificities.
………..and the world of discoveries carried on till date and will carry on till man is there.
PLASMID DNA
A gene is a part of a chromosome & is responsible for some character or trait of an organism or cell. It is made of DNA. 1953, it was universally accepted that DNA is the genetic substance of most microorganisms & higher organisms.
Highly purified DNA has been found to be complex macromolecular or polymeric chemical compounds that contain four kinds of smaller building blocks called deoxyribonucleotides. Each deoxyribonucleotide is made up of three components – a phosphate, a pentose sugar called 2-deoxyribose & Pyrimidine & Purine nitrogenous bases. In 1953 Watson & Crick gave the double stranded structure model of DNA.
A plasmid is a DNA molecule, other than the bacterial chromosome, that is capable of independent replication & transmission or plasmid may be defined as autonomous element, whose genomes exist in the cell as extra chromosomal unit. In 1952 Toshua Lederberg introduced the word “Plasmid” & he defined it as an extra chromosomal genetic element. The size of plasmid vary from 1Kb to more than 200Kb, most of them are double stranded, covalently closed, circular molecules that can be isolated from bacterial cells in a super helical form.
Plasmids are found in a wide variety of bacterial species. Most plasmids have a narrow host range & can be maintained only in a limited set of closely related species. These behave as accessory genetic units that replicate & are inherited independently of the bacterial chromosome.
There are several types of bacterial plasmids, but the three widely studied types are:
1. F-plasmids – these are responsible for conjugation2. R-plasmids – carry genes for resistance to antibiotics3. Col-plasmids – these codes for colicins, the protein
that kills sensitive & coil cells, they also carry genes that provide immunity to the particular colicin.
Each plasmid is maintained in the bacterial cells at a characteristics copy number (the copy number of plasmid is defined as the average number of plasmids per bacterial cells or per chromosome under normal growth condition) mainly due to its replication system. In these respect the plasmid are of two types:-
1. Single copy : The replication control of single copy plasmids is the same as that of their bacterial host cells so that they replicate and aggregate with the bacterial chromosome, this is called stringent replication.
2. Multiple copy : The replication control of multiple plasmid is different from that of their bacterial host genome so that they undergo more than one replication for each replication of their host genome. This is referred to as relaxed condition. They have evolved a variety of mechanism to maintain a stable copy number of the plasmid in their bacterial host and to partition plasmid molecules accurately to daughter cells. Plasmids are dependent to a greater or lesser extend on the enzymes and protein encoded by their host for their replication and transcription. Plasmids frequently contain genes coding for enzymes that are advantageous to the bacteria host. These genes specify a remarkably divorce set of traits, many of which are of great medical and commercial significance. Among
the phenotypes conferred by plasmids are resistance to production of antibiotics, degradation of complex organic compounds and production of colicins, entrotoxins and restriction and modification enzymes.The plasmids under relaxed control replication permit their accumulation in very large number. These are the plasmids which are used as cloning vectors, due to their increased yield potential.
One of the standard cloning vectors widely used in gene cloning experiment is pBR322, which is 436bp DNA and was derived from E.coli plasmidin colEI. PBR322 was named after Bolivar and Rodriguer who prepared their vector. It has genes for resistance against two antibiotics,(tetracycline and ampicillin), an origin of replication of a variety of restriction sites for cloning of restriction of fragments obtained through cleavage with a specific restriction enzyme. Another vector pBR327 was derived from pBR322. Both pBR322 and pBR327 are common plasmid vectors.
Schematic Representation of Pbr 322
Plasmid Isolation:In plasmid isolation, the elimination of chromosomal DNA
forms an important step. The chromosomal DNA will always be attached to the cell wall. A good method is the one that allows very small breakage of the chromosomal DNA. Both the chromosomal and plasmid DNA are circular in shape. During the cell extract preparation of chromosomal DNA breaks into linear forms the separation of linear and circular molecules result in pure plasmid (circular). In E.coli and other related species controlled lysis (EDTA+glucose) is advised lysozyones & EDTA are used in combination or alone to break the cell wall of bacterial. A very gentle cell disruption prevents whole cell breakage of the divalent metallic ions that are essential for preserving the overall structure of the envelope of the cell and to inhibit cellular enzymes that could degrade DNA. EDTA with lysozymes with Sucrose prevents
pBR 322 Ampicillin Resistance site
Origin of Replication
Tetracycline Resistance Site
Eco RI
cell burst in straight way. Instead a sphearoplast (bacterial cell with partially cell wall less that retain an intact cytoplasmic membrane) is form. SDS induces cell lysis rapidly. This causes very little breakage of bacterial DNA. Most plasmid exists in the cell as supercoiled molecules. Addition of NaOH increases the cell extract pH to 12 – 12.5 that cleaves the hydrogen bonding in non-supercoiled DNA molecules causing double helix to unwind & the two polynucleotide chains to separate. Once the acid is added the denatured DNA reaggregates into a tangled mass that settles down during centrifugation leaving only plasmid in the supernatant. Addition of sodium acetate or potassium acetate add SDS removes most of the protein and RNA by making them insoluble. Phenol and RNAase addition totally removes the residual protein and RNA contamination. SDS and ether addition removes the lipids and similar substances available in the extract. Addition of chilled ethanol precipitates plasmid in the pure form.
Different methods of isolation of plasmid DNA – There are many methods available to isolate plasmid from bacteria. These methods include 3 major steps –
1) Growth of the bacterial culture2) Harvesting in lysis of bacteria3) Purification of plasmid DNA
The methods of isolation are based on the several physical differences between plasmid DNA and bacterial DNA, the most obvious of which is size. The largest plasmids are only 8% of the size of E.coli chromosome and most are much smaller than this. Techniques that can separate small DNA molecule from large ones should therefore effectively purify plasmid DNA.
The different methods of isolation are:
1. Separation on the basis of size : The usual stage at which size fractionation is performed is during preparation of the cell extract. If the cells are lysed under very carefully controlled condition only a minimal amount of chromosomal DNA breakage occurs. The resulting DNA fragments are still very large – much larger than the plasmid and can be removed from the cell debris by centrifugation. This process is aided by the fact that the bacterial chromosomes physically attached to the cell envelope, so fragments of the chromosome sediments with the cell debris of these attachments are not broken.
2. Separation on the basis of conformation : Before considering the ways in which conformational differences between plasmids and bacterial DNA can be used to separate the two types of DNA. It is not strictly correct to say that plasmids have a circular conformation because double stranded DNA circles can take up one of two quite distinct configurations. Most plasmid exists in the cell as supercoiled molecules. The supercoiled conformation can be maintained only if both polynucleotide strands and intact, hence the more technical name of ‘covalently closed DNA (CCC)’. If one of the polynucleotide strands is broken the double helix reverts to its normal relaxed state and the plasmid takes on the alternative conformation called open circular (OC).
3. Alkali lysis method : The basis of this technique is that there is a narrow pH range at which non-super coiled DNA is denatured, whereas super coiled plasmids are not. Alkaline lysis in combination with the SDS has been used more than 20 years to isolate plasmid DNA from E.coli.
Ethidium Bromide : Cesium chloride density gradient configuration :-This is a specialized version of the more general technique of equilibrium or density – gradient centrifugation. Ethidium Bromide – cesium chloride density gradient centrifugation is a very efficient method for obtaining pure plasmid DNA when a cleared lysate is subjected to the procedure, plasmid bond at a distinct point separated from the linear bacterial DNA, with the protein floating on the top of the gradient and RNA pelleted at the bottom. Shining ultraviolet radiation on the tube, which causes the bond EtBr to fluorescence, can see the position of the DNA bands. Puncturing the side of the tube and withdrawing a sample with a syringe remove the pure DNA. The EtBr bound to the plasmid DNA is extracted. With n-butanol and the CsCl removed by dialysis. The resulting preparation is virtually 100% pure and ready for use as cloning vehicle.
RESTRICTION ENDONUCLEASE
Endonuclease are enzymes that produce internal cuts, called cleavage in DNA molecules. Werner Arber postulated the presence of restriction enzymes during 1960s. while the first true restriction endonuclease was isolated in 1970 by H.O.Smith.
Type-2 endonuclease are commonly described as restriction enzyme. It generates reproducible nucleotide fragments from specific DNA. They cleave double stranded DNA by hydrolyzing two phosphodiester bonds within defined nucleotide sequences.
The name of restriction enzyme is derived from genus and species and bacterium from which it is isolated. The first letter of the genus name and the first two letters of the species are combined to form the enzyme name. This is followed by a strain designation if needed. In many instances a bacterial strain contains more than one restriction endonuclease. When this occurs each enzyme is assigned a roman numeral. Thus Bam HI was 1st enzyme activity reparted from Bacillus amyloliquefaciane strain H.
There are 3 distinct types of restriction endonucleases:-
Type I – These are complex endonucleases and have recognition sequences of about 15 bp. They cleave at about 1000 bp away from their recognition sites. Eg. Ecok, EcoB etc.
Type II – These are remarkably stable and induce cleavage either in most cases within their recognition sequences or very close to them. They require Mg ++ ions for cleavage. The 1st type 2 enzymes to be isolated were HindII in 1970. Only type II restriction endonuclease is used for restriction mapping and gene cloning.
Type III – these are the intermediate between the type I and type II enzymes, they cleave DNA in the immediate vicinity of their recognition sites (24-26 bp away from their recognition sites) eg. EcoPI, EcoPI5 etc.
A given sequence in DNA is recognized and can be cleaved by more than one restriction enzyme. The term “isoschizomer” describes a group of enzymes that recognize the same sequence in DNA eg. Sma I and Xma I.
The recognition sequences for type II endonuclease form palindromes with rotational symmetry meaning, the base sequence in the first half of one strand of a DNA double helix is the minor image of the second half of its complementary strand thus, in such palindromes, the base sequence in both strands of DNA duplex reads the same when read from the same end of both the strands.
ECORI recognition site: 5’ GAATTC 3’
3’ CTTAAG 5’
Most of the type 2 endonucleases have recognition sites of 4, 5 or 6 bp that are predominantly GC rich.
Some restriction enzymes cut symmetrically (exactly in the center of the recognition site) and generate blunt end or flush
ends. So while most enzymes produce staggered cuts in which the two strands of a DNA double helix are cleaved at different locations this generates protruding ends. Due to the palindromic nature of the target sites, the 2 protruding ends generated by a cleavage enzyme have complementary base sequence. As a result they readily pair with each other, such ends are called cohesive or sticky ends.
The DNA from bacteriophage λ is the widely used substrate for screening restriction enzymes because it is often difficult to determine a characteristic pattern from a lambda digest. Smaller DNAs, such as the replicated form of bacteriophage ɸX174 and SLVO are also used as substrates.
It is important to note that enzymes do not attack the DNA of the cell in which they are formed. It is because a parallel series of enzymes exist that modify the DNA in such cells by adding extra methyl group of the bases within the recognition sequence. Such sequences are then marked as self and are not subjected to attack.
Significance of restriction endonucleases :-
Among all the restriction enzymes restriction endonucleases have immensely contributed towards our understanding of gene structure and function. The DNA fragments produced due to cleavage of small genome by restriction enzyme can be used for wide range of genetic manipulation. The main applications of restriction endonucleases include physical restriction, construction, sequencing of large DNA genomes, construction of gene libraries and molecular cloning.
EXAMPLES OF RESTRICTION ENZYMES
Enzyme Organism from which derived
Target Sequence(Cut at *)5’ 3’
Ava I Anabaena Variabilis C*C/TCGA/GG
Bam H I Bacillus amyloliquefaciens
G*GATCC
Bgl II Cillus globigii A*GATCT
Eco R I Escherichia coli Ry 13
A*AATTC
Eco R II Escherichia coli R2 45
*CCA/TGG
Hae III Haemophilus aegyptius
GG*CC
Hha I Haemophilus haemolyticus
GCG*C
Hind III Haemophilus influnzae Rd
A*AGCTT
Hpa I Haemophilus parainfluenzae
GTT*AAC
Kpn I Klebsiella pneumonia GGTAC*C
Mbo I Moraxella Bovis *GATC
Pst I Providencia sturatii CTGCA*G
Sma I Serratia marcescens CCC*GGG
Sst I Streptomyces Stanford
GAGCT*C
Sal I Stretomyces albus G G*TCGAC
Taq I Thermophilus aquaticus
T*CGA
Xma I Xanthamonas malvacearum
C*CCGGG
LIGATION
The enzyme DNA ligates is used to covalently link the 3’ – OH terminal of one strand of the 5’ – phosphate end of a second strand. Thus the enzyme can repair single stranded breaks such as those present in hydrogen bonded sticky ends of two DNA preparation that have been cleaved with the same restriction enzyme DNA ligase from T4 and higher organism utilize ATP as cofactor. In addition to repairing single stranded breaks as does E.coli enzyme, T4 ligase can link RNA and DNA strands aligned on a complimentary DNA template. Another enzyme RNA ligase joins two single stranded DNA or two RNA molecules or RNA molecules to single stranded DNA molecule (seigeno et al, 1977).
During the process of ligation generally the vector anneals back in the original form, which decreases the percentage of real recombinant molecules formed. Linkage of two DNA molecules is favored by a high concentration of DNA molecules. Therefore, after ligation of DNA segments at high concentrations of DNA, the mixture is sometimes diluted so that the circularization decreases as the length of the DNA segments increases. Due to this reason, short DNA segments are cloned more easily than the long ones.
Sgaramella et al (1970) reported that the ligase enzymes can join (blunt end) fully base paired DNA duplexes. Although blunt end joining occurs to a much slower rate as compared to sealing of single strand nicks, it is of immense importance especially for joining DNA molecules that lack cohesive ends. DNA segments obtained by shear by using two different restriction enzymes or by using or restriction enzymes that produce blunt ends, can be joined without adding homopolymer tails. Blunt end ligation of longer molecules obtained by shearing DNA at random than at specific sequences offers the possibility of cloning intact genes.
5’ --------------3’ 5’ CCGAATTCGG 3’3’ --------------5’ 3’ GGCTTAAGCC 5’Blunt ended Linker SequenceDNA fragment
T4 Ligase
5’ GGCTTAAGCC -------------------------------- GGCTTAAGCC 3’3’ CCGAATTCGG -------------------------------- CCGAATTCGG 5’
Linker DNA
Linker
Fragment
Eco RI
GCC --------------------- GGCTTAA
AATTCGG --------------- CCG
DNA fragment with
Eco RI cohesive ends
AGAROSE GEL ELECTROPHORESIS
Alkaline agarose electrophoresis was developed in Bill Studiers laboratory at Brookhaven National laboratory as a replacement for laborious alkaline gradient centrifugation of bacteriophage T7 DNA.
Agarose is a polysaccharide made up of repeating residues of D & L galactose joined by α(1→3) & β(1→7) glycosidic linkages. The α galactose residue has an anhydro bridge between the three and six positions. Chains of agarose form helical fibres that aggregate into super coiled structure with a radius of 20 – 30 nm and has a molecular mass of 120 KD.
Structure of Agarose
D-Galactose 3, 6 anhydro α - galactose
Factors affecting the rate of migration through agarose gel : -
Several factors determine the rate of migration of DNA through agarose gels.
1. The molecular size of the DNA :
Molecules of double stranded DNA migrate through gel matrices at rates that are inversely proportional to the log10 of the number of base pairs. (Helling et al 1974). Larger molecules migrate more slowly because of greater frictional
drag and because they worn their way through the pores of the gel thus efficiently than smaller molecules.
2. The concentration of agarose:
A linear DNA fragment of a given size migrates at different rates through get containing different concentration of agarose. There is a linear relationship between the logarithm of electrostatic mobility of the DNA (u) and the gel concentration (i) i.e., describe by the equation
Log u = log uo-krl
Where u = is the free electrophoretic mobility of DNA and kr is the retardation coefficient constant related to the properties of gel and size and shape of the migrating molecules.
3. The conformation of the DNA :
Superhelical circular (form I ) nicked circular (form II) and linear (form III) DNA’s migrate through agarose gels at different rates (Thorne 1966-67). The relative mobilities of the three forms depend primarily on the concentration and the type of agarose used to make the gel, but they are also influenced by the strength of the applied current, the ionic strength of the buffer, and the density of superhelical twists in the form I DNA (Johnson and Grossman1977).Under some conditions, form I DNA migrate faster than form II DNA, while under other conditions, the order is reversed.
4. The presence of the ethidium bromide in the gel and electrophoresis buffer : Intercalation of ethidium bromide causes a decrease in the negative charge of the double stranded DNA and on increase in both its stiffness and length. The rate of migration of the linear DNA dye complex through gels is consequently retarded by a factor of
5. The applied voltage :
At low voltages, the rate of migration of linear DNA fragment is proportional to the voltage applied. However, as the strength of electric field is raised, the mobility of high molecular weight fragment increases differentially. Thus, the effective range of separation in agarose gel decreases as the voltage is increased to obtain maximum resolution of DNA fragment > 2kb in size, agarose gel should be run at no more than 3-8 V/cm.
6. The electrophoresis buffer :
The electrophoretic mobility of DNA is affected by the composition and ionic strength of the electrophoresis buffer. In the absence of ions, electrical conductivity is minimal and DNA migrates slowly, if at all in buffer of high ionic strength electrical conductance is very efficient and significant amount of heat is generated, even when moderate voltage are applied. In the worst case, the gel melts and DNA denatures.
7. Gel loading buffer or dye :
Gel loading buffer is mixed with the samples before loading into the slots of the gel. There buffers serves three purposes. They increase the density of the sample, ensuring that the DNA sinks evenly into the well, they add color to the sample, thereby simplifying the loading process; and they contain dye that in an electric field, move towards the anode at predictable rates. Bromophenol blue migrate through agarose gel 2-2 fold faster than xylene cyanol FF, independent of the agarose concentration. Bromophenol blue migrates through agarose gels run in 0.5x TBE at approximately the same rate as linear double – stranded DNA 300 bp in length, whereas xylene cyanol FF migrates at approximately the same rate as linear double-stranded DNA 4kb in length. These relationship are not significantly affected by the concentration of agarose in the gel over the range of 0.5 – 1.4%
REFRIGERATOR
It is an instrument used to keep the stock solution of media and other materials used for culturing vegetables, fruits etc. This is to prevent contamination by microorganisms by way of cold atmosphere.
CONSTRUCTION:
A refrigerator has the following parts: -
THROTTLE VALVE:- A mechanical container, which changes the face or pressure
of a working fluid. The most commonly used refrigerant or working fluid is choler floor carbon.
EVAPORATOR: - Area where the materials are kept. Here the working fluid gains
the heat and is transferred into vapor.
CONDENSER: - It is a heat exchanger where the working fluid gives out the heat.
WORKING PRINCIPLE:The working fluids entertain the evaporator area. When the liquid comes in contact with the vessel it absorbs heat from the vessel. The liquid transferred into vapor. The hot vapor reaches the throttle valve, where it is condensed into liquid again. The cycle is repeated.
USES:It is used to keep all materials used for culturing, free of contamination by microbes.
pH METER
PRINCIPLE:
The pH meter is provided with an extremely stable D.C amplifier with high input in pH for use with high resistance calamel electrode. It has a built in voltage stabilizer, which is helpful in avoiding effective changes in voltage supply. The electrode, detect change in hydrogen ion concentration and the differences in electrical potential is detected by the potentiometer which expresses the pH on a digital display.
PROCEDURE:
CALIBRATION:
The pH meter is switch on and allowed to warm up for fifteen minutes without any disturbance. The electrode is washed with distill water and wiped dry with filter paper. The temperature adjustment knob is used to set the instrument to room temperature. The electrode is then rinsed with small amount of pH 4 buffer solutions. The washings are collected as waste. The buffer of pH 4 is taken in a 50ml beaker and the bulb portion of the electrode is immersed into it carefully. The selector knob is put to pH position. If the display does not read for using the calibration knob the pH display is adjusted to 4. Wait for 30 seconds till the value
remains constant. The selector knob is set back to stand by the position. The buffer is removed and the electrode is then rinsed with pH 9.2 buffers. The washings are collected as waste. The bulb portion of the electrode is immersed in the breaker containing the buffer. The selector bulb is put in the pH position. If it does read 9.2 it is re-calibrated to do so. Wait for 30 seconds. The selector, switch is set back to stand by position. The standardization is now complete and instrument is ready to use.
DETERMINATION OF pH OF THE GIVEN SAMPLE:
A 100ml beaker is rinsed with distilled water. The electrode is rinsed with distilled water. The electrode is wiped dry using filter paper and is then immersed in the test sample taken in 100ml beaker after mixing. The selector knob is put to pH position and the pH is taken down from digital display after value becomes constant. The electrode is removed from the sample and rinsed. With distilled water, wiped dry and kept dipped in the beaker containing distilled water.
MICROSCOPE
Light microscope is one where glass lenses are used and visible light is used as the source of light. The most commonly used optical microscope is known as compound optical microscope. Here, the microscope field appears bright and the objects being stained appear darker. The different parts of a microscope are:-
METAL STAND:It is a supporting stand consisting of the followings:-
A broad base or foot.A short pillar attached to the base.An upright curved arm attached to the pillar.
The stand serves to give stability to the instrument.
LIGHT SOURCE:
It is situated at the base. It is a strong light source either consisting of a built in electric lamp or a mirror which can be adjusted so as to maximum light falls on the object being viewed. The mirror has one plane and one curved surface.
DIAPHRAGM:
This is attached to the pillar above the light source. It is adjustable and regulates the amount of light entering the condenser.
CONDENSER:
It is present above the pillar or above the diaphragm which concentrates the light rays on the object.
STAGE:
It is a horizontal platform that holds the microscope slide in position. It is a 3 to 4 inch square or circular platform width and opening in the Centre to permit light from the condenser to fall on the object.
BODY TUBE:
It is attached to the curved arm of the microscope. It may contain a system of prism and reflectors. At the lower end of the body tube is objective lens system and at the top end is the circular lens system or eye piece, usually containing two or three lenses.
OBJECTIVE SYSTEM:
These are primary lenses that magnify the specimen giving a dually enlarged image. The three types of objectives are :-Low power lens:-It has a focal length of 16mm and 2/3rd inches and magnification of 100x.
Oil power lenses :-It has a focal length of 2mm or 1/12th inches and magnification of 100x. These objective lenses differ from the low power objective lens and the high power objective lens is having higher magnification and is made of views. Oil immersion lens is always used with some special oil like cedar wood oil. The image obtain has a higher magnification and better resolution. The oil that is used should have refractive index close to that of the glass.
DOUBLE DISTILLATION UNIT
This is an instrument used for distillation of water. It is available in a fully glass or quarts setup in either cylindrical form or as round bottom flasks having long necks and quarts heating coils and steam condensing unit. Two separate round bottom flasks are used in order to obtain single and double distilled water.
In a vertical type setup, there are two main units made up of quarts. The first unit is lower in position and has a boiler with a heating coiled arrangement. It has an inlet for water which has a connection to a work source and an arrangement for excess water to be drained out. The second unit is upper in positioned has a boiler with a heating coiled. This is connected to the lower unit by a quarts tube arrangement internally for the entry of steam from the boiler of unit-I. There is cooling jacket outer to the steam collection unit. It is again connected to a water source. This upper unit has an outlet for the flow of double distilled water.
Water flows from the source into the first boiler and forms water vapour on heating which enters the second boiler. Here it is condensed and accumulates and is again subjected to boiling. The water vapour formed here is condensed due to a cooling jacket and the water, which is now double distilled, pours through the outlet. A water level electrode is filled with both units in order to control switching on and off of the unit automatically.
PRECAUTION:The water that has to be distilled should always be demineralized to prevent
salt deposition on the heating coil. Continuous supply of cool tap water must be ensured in order to account for the continuous running of the unit.
MONOPAN BALANCE
INSTRUCTION:The Monopan balance should be kept in the Balance room free from any
vibration, air thrust, strong ray of light, suspended dust particles, acid and chemical fumes, strong heat etc. An air-conditioned room is preferable for this purpose. If any fan is put on, it should be put off during the operation of the balance. Fluorescent light is well suited in the laboratory or room for fair readings of the projection scale. The platform for projection installation should be, as far as possible, if this three point electrical socket bearing source of 220/230 volts a/c supply should be very near the reach of the platform base.
DESCRIPTION:
The Monopan balance is a balance, which has only one pan onto which weighing substances are placed. The pan is used for weighing is hung to the hook provided just in the middle of the glass chamber. The glass chamber has two sliding glass doors, one on each side to carry on the operation. Weights have to be
calibrated using the weights provided. There are also different knobs provided for adjustment.
Clean the external part of the balance and also the weighing pan using a soft duster in order to remove the dust particles. Hang the weighing pan to the hook provided at the middle of the glass chamber, at one end of the beam, the micro glass slides are fitted in a circular hole and the other end pan is supported by preloaded ring weight attachment. A leveling bulb with an etched circle on its round flat glass is fitted on the right side of the upper plate. It is essential to level the balance with the help of leveling leg screws and the leveling bulb. Rotate the screws one at a time, which makes the balance body go up or down until the bulb comes into the etched circle. The bulb in the circle indicates perfect leveling of the balance ring weights in their own position. The touching of the body with the ring weights is a common during transit. A brief account about knobs:
a. Operating knob: A molded triangular shaped knob with a slotted hole
b. Weight knob: Molded tapes type rounded knob with plated brass insert into it.
c. Zero adjusting knob: This is similar to the weight knobs.
d. Digital knobs: A flat type rounded knob with a solid brass insert in it.
After all the parts are fixed, put on the three-pin plug in the three-pin socket and switch it on. Now you will see that the pilot lamp indicates the flow of
electricity in the transformer of the balance. The pilot lamp is placed on the wall of the glass chamber.
Keep all the weights in zero position by rotating the weight knobs and digital knobs. Move the operating knob quarter of a circle clockwise and look at the vernier screen glass. If the zero of the glass scale then, it is all right. But if it is does not differs by 1 or 2/3 division, then it can be made to coincide by moving the zero knob fitted in the right side to the top. Now the balance is ready for working.
CENTRIFUGE
The centrifuge is an instrument used to separate substances of different density suspended in the solution.
The rate of settling of particles depends upon the density and radius of the particles and upon the viscosity and density of the fluid where the particles are suspended.
PRINCIPLE OF WORKING:
The material to be centrifuged is taken in the tube and placed inside the centrifuge in pairs and balanced accurately. The tubes must be placed diagonally opposite each other. A balancing tube having water can be used if an odd number of tubes are present for centrifugation. The lid is closed and the centrifuge is adjusted to the required temperature and r.p.m. Then the centrifuge is switched on.
PRECAUTIONS:
i. The tubes must be kept in pairs.ii. Take care to lubricate periodically.
iii. Close the lid properly.iv. Bring the restore to zero.v. Do not stop the motor by applying force.
ELECTROPHORESIS UNIT
Electrophoresis is an analytical tool by which biochemists can examine the movement of changed molecules in an electric field. Modern electrophoretic techniques use a polymerized gel like matrix as a support medium. The sample to be analyzed is applied to the medium as a spot or thin band, hence the term “Zonal” electrophoresis.
The migration of the molecules is influenced by the applied electric field, the rigid, maze like matrix of the gel support and the size, shape, charge and chemical composition of the molecules to be separated in electrophoresis, which is a relatively rapid and convenient technique, is capable of analyzing the purifying several different types of biomolecules, but especially proteins and nucleic acids. Although it is difficult to provide accurate theoretical description of the electrophoretic movement of the molecules in a gel support, zonal electrophoresis has gained widespread use in purifying and identifying biochemical and in determining their molecular sizes. The electrophoresis apparatus consists of the separating medium connected to two electrode tanks by means of filter paper or gaze pads. The electrodes are divided into two compartments connected by cotton wool wicks. One part contains the platinum electrode and the other is in contact with the electrodes even in a buffered solution; the division of electrophoresis apparatus into two compartments ensures that such chances close to the support phase are minimized. The concentration between this phase and the buffer solution is made by several thickness of watmann 3nm filter paper or hospital gauze saturated with buffer solution. This concentration should be such as to cause a minimum drop in potential across its length.
LAMINAR AIR FLOW
INTRODUCTION:This is the most recent, suitable experiment and reliable system for all types of aseptic preparation, manipulations and transfer works. It is an absolute requirement for plant tissue culture works. It enables one to work openly, easily and for long period which is not possible inside an inoculated chambers.
CONSTRUCTION:It consists of a main box or cabinet with an upper and a lower part. In the lower part there is a blower motor with blower fitted in the outlet opening to the upper chamber. The front and back of the lower chamber is in continuous with an air pre-filter to keep dust and fine particles away from entering the path and the chamber. The upper chamber is fitted with an air tight HEPA filter [0.2-0.4µm]
WORKING PRINCIPLE:
The main principle of the LAF is to provide asceptic condition. It works on the principle air filtration with the help of motor is nuked by bower entering filtered in the lower chamber and blow under pressure to the upper chamber where it is forced to pass the HEPA filters having pore size of 0.1-0.4 µm to blow air to the working area. It continuously sucks air and blows out at uniform velocity and in parallel flow lines.
PRECAUTION:
1) The LAF should always be kept in a closed and dust free place.2) Before using LAF the UV light should be put on for half an hour to kill
any microorganisms on the surface.3) The blockage of the lower filter has to be checked. So also the pressure
inside and leakage from the HEPA filter.4) The surface of the LAF must be swabbed with 70% ethanol before and
after use.5) Sterile medium and instruments should be kept in the LAF.6) Before starting work in the LAF hood, the hands should be cleaned
with 70% ethanol.
As soon as the outer door of the working area is opened, the air flow should be put on.
INCUBATOR
INTRODUCTION:
Incubator is an apparatus used for the cultivation of microorganism at constant temperature and humidity. It consists of a rectangular box with a double wall insulated with asbestos. It is heated by an electric current and maintained at constant temperature by means of thermometer. The incubator is provided with a double door, the inner one of a glass and outer one is the same material rest of the chamber. The bacterial culture is generally maintained at 37˚c (±2).
It is rectangular or square made up of steam and provided with doors that are in a glass door, outer main door. The main door is doubled layered with asbestos lining which acts as an insulator when the door is closed. The inside of the incubator is provided with removable perforated racks. It is also provided with foil. The incubator also has knobs to regulate the temperature and heat. There is thermometer fix which indicates temperature.
PRINCIPLE:
Any successful isolation or culturing depends on good incubator. It provides the optimum temperature for growth. The incubator maintains constant temperature during the growth of an organism. Different organisms require different temperature ranges which permit their proliferation.
WORKING:
Air is let in where a fan circulates the air. When the desired temperature is reached the temperature us maintained with the help a thermostat. The heating capacity in an incubator ranges from 25˚c to 110˚c
USES:
Incubator is used to provide optimum temperature for the growth of different organisms.
ISOLATION OF PLASMID DNA BY ALKALI LYSIS METHOD
AIM:
The objective of this module is to isolate plasmid DNA from the bacterial host cell and to learn basic techniques and procedures used in rDNA technology by alkali lysis method.
PRINCIPLE:
Plasmid DNA may be isolated from bacterial culture by treatmrnt with alkali and Sodium Dodecyl Sulphate (S.D.S). Alkaline lysis in combination with the S.D.S, has been used for more than 20 years to isolate plasmid DNA from E.coli. Exposure of bacteria suspensions to the strongly anionic detergent at high pH opens the cells wall, denatures chromosal DNA and proteins and released plasmid DNA into the supernatant. Although alkaline solution completely descipates base pairing,through strands of closed circular plasmid DNA are unable to separates from each other because they are topologically interwined. As long as the intensity and duration of exposure to OH- is not too great, the two strand of plasmid DNA falls once again into register when the pH is return to neutral.
During lysis, bacterial proteins broken cell walls and denatured chromosal DNA becomes enmeshed in large complex that are coated with dodecyl sulphate. These complexes are efficiently precipitated from solution when sodium is replaced by potassium ions. After the denature material has been removed by centrifugation native plasmid DNA can be recovered from the supernatant.
REQUIREMENT:
Chemicals required:
L.B BrothSolution 1: 20mM glucose, 25Mm Tris HCL, 10 Mm EDTA.Solution 2: 0.2N NaOH, 1% SDSSolution 3: 5M potassium acetate, glacial acetic acid.Solution 4: Ethanol (100%) T.E buffer: 10mM Tris HCL and 1 mM EDTAEtBrTAE
Role of chemicals:
1. Solution 1:It has three components -
Glucose --- it gives osmoregulatory effectTris --- it is trihydroxy methyl amino methane and it is an anionic base.HCL is an acidTris HCL is used to maintain pH.EDTA --- It is a chelating agent. It chelates the divalent metallic ion (Mg++). The main function of solution 1 is to keep the cells intact because of which spheroplast remain alive. Hence, it gives an osmoregulatory effect.
2. Solution 2:It has two components – NaOH – it denatures genomic and plasmid DNA.SDS (Sodium Dodecyl Sulphate) – it is a detergent and it cleaves lipid, protein and other cell components.
3. Solution 3:
It has two components –
Potassium acetate and glacial acetic acid .
It acts as a neutralizing solution as it lower the pH of DNA.
4. Solution 4:
It consists of 100% ethanol or propane. Ethanol precipitates the DNA as it intercalates between the bases of DNA fragments and helps them to precipitates down.
5. TE buffer:It is added to dissolve the DNA in the solution for gel electrophoresis.
6. EtBr Buffer (Ethidium Bromide): It provides fluorescence to the plasmid DNA under UV space. Light, as Ethidium Bromide to DNA molecules by interacting between adjacent base pairs causing partial unwinding of double helix.
7. Gel Loading dye:It consists of mainly Bromo Cresol Blue or Bromo Thymol blue or Bromo Phenol blue and glycerol. Bromo cresol or bromo thymol or bromo phenol blue gives color to the gel. They are also called as indicator dye. They are more negatively charged than DNA. Therefor it moves ahead DNA during gel electrophoresis.
Glycerol is very viscous. It gives density to the plasmid DNA to get settled in the well, as the plasmid DNA is very light.
8. T A E:
Tris Acetate EDTA is the running buffer.
MATERIAL REQUIRED:
Beaker Test Tube Vials Conical Flask
INSTRUMENTS REQUIRED:
Micropipettes Electrophoresis Apparatus Transilluminator Centrifuge Vortex
PROCEDURE:
1.5 ml of culture was pipetted out in a 1.5 ml tube.
The cell was spin down at 6000 rpm for 8-10 minutes. The supernatant was then discarded. Keeping the vial inverted on the blotting paper drained the soup. The vial was then kept in ice.
The cell was resuspended in 100µl (0.1ml) of ice cold solution 1 to get uniform suspension or vortex gently. It was then kept on ice for 5 minutes and shifted to room temperature.
Solution 2 was thawed and 200µl (0.2ml) of solution 2 was added. The tube was tightly close and the content was mixed by inverting the tube 5-6 times (do not vortex). Solution was added to room temperature.
150µl (0.15ml) of solution 2 was added. T tubes were tightly closed and the contents were mixed gently by inverting the tubes. Then kept on ice for 5 minutes. (the temperature should exceed 5 minutes because it may renature the genomic DNA).
It was spinned at 6000-8000 rpm for 10 minutes.
The supernatant was immediately transferred to a fresh vial and 450µl (0.45ml) of solution 4 was added inverting to precipitate the DNA mixed. It was kept at room temperature for 10-15 minutes.
The sample again centrifuged for 20 minutes at 10000 rpm. The supernatant was decanted and stand the tube in inverted position on paper to allow the fluid to dry away. The DNA was seen as white precipitate sticking to the side. It was dried till there was no trace of solution 4 at 37 ˚c for 10 – 15 minutes.
20µl of XTE was added from the side and gently tapped so that the DNA goes into the solution.
RESUTL AND OBSERVATION:
Genomic DNA isolated from given plants leaves, an electrophoresis produces a gel under the UV transilluminator as:
The lane with genomic DNA produces a streak of bands. Another thin band was visible ahead of tracking dye.
DISCUSSION:
The methodology of alkali lysis with SDS extracts plasmid DNA from E.coli by breaking open the cell wall and forming sphereoplasts initially. On centrifugation the plasmid DNA is separated from the chromosomal DNA, based on size, where the plasmid DNA is found in supernatant.
Addition of other chemical like phenol, chloroform, alchohol etc. purifies the plasmid DNA from other components of bacterical cells. Finally the plasmid DNA is precipitated using absolute alcohol.
The electrophoresis result of the isolated plasmid DNA on agarose gel can be explained as
The three bands in lane 1 are due to linear, nicked and supercoiled plasmid DNA. Since the conformation of each of these form varies, there electrophoretic mobility also differs and leads to three bands. The mobility decreases in the order CCC supercoiled, open circle (nicked) and linear. Hence the band, which has moved further from the well, is due to super coiled plasmid DNA, while the band close to well is the linear plasmid DNA.
ISOLATION OF GENOMIC DNA FROM PLANT
AIM: To isolate the total genomic DNA from plant.
PRINCIPLE: The principle behind the experiment is to isolate chromosomal DNA from plant leaf by the process of ethanol precipitation. In eukaryotic cell chromosomal DNA is found in the nucleus. It doesn’t exist as a free molecule but as the complex association of DNA, RNA and protein. Negatively charged DNA is associated with numerous positively charged basic proteins. This include enzymes for replication, transcription and recombination, repair as well as proteins for unwinding super coiled DNA. By regular precipitation of cellular organelles and other matrix only DNA is isolated and it is precipitated by ethanol or sodium citrate and thus can be as white threads.
REQUIRMENTS:
A)Chemical Requirments:- Lysis buffer (10mM Tris, 5mM EDTA, 0.5% SDS), Phenol chloroform (1:1), isopropanol and chloroform (24:1), sodium acetate, plant material, ice cold ethanol, distilled water.
B) Apparatus Required: - Incubator, electronic balance, centrifuge, test tubes.
PROCEDURE:
Weigh 250 gms of the plant tissue and dispense into the vessel. Add 1 to 2 ml of the lysis buffer.
Centrifuge the homogenase at 3000 rpm for 8 mins. Collect the supernatant and add equal volumes of phenol chloroform. Centrifuge at 3000 rpm for 5 mins. Collect the aqueous phase without
disturbing the interphase. To it add equal volumes of chloroform isoamyl. Invert gently and centrifuge
at 3000 rpm for 10 mins. Collect the aqueous phase and add 1/28th of 3M sodium acetate. Add double the volume of ethanol to it.
RESULT AND OBSERVATION:
After homogenization process chloroform was added and centrifuged. The centrifugation resulted in 3 layers:
1. The bottom most was chloroform layer containing protein and carbohydrates.
2. Middle layer was of cell debris.3. The top most layers was containing DNA.
The addition of ice cold ethanol to the DNA containing supernatant bringing about precipitation of DNA. DNA floated on the top like cloud. Cold condition aid in keeping the DNA intact. treatment with ethanol and centrifugation was repeated to get a clear DNA
precipitate, which was later dried and dissolved in TAE buffer overnight.
The sample was run on an agarose gel to visualize the DNA.
Genomic DNA isolated from goven plant leaves on electrophoresis produced a gel under UV transilluminator as:-
The lane with genomic DNA produces a streak of band. Another thin band was visible ahead of tracking dye.
DISCUSSION : By regular precipitation and breakage of cellular organelles, the DNA is isolated. During this process, different reagents used acts or reacts differently on the cellular organelles such as –
EDTA – It chelates divalent cations like magnesium. The removal of such ions vacant plasma and nuclear membrane making them easier to lyse.
The removal of these ions also inhibits DNA digestion by DNase enzyme released during homogenization.
SDS – It is a detergent, which emulsifies the plasma and nuclear membrane promoting lysis SDS is also required by denaturing the proteins.
SODIUM ACETATE – It increases the ionic concentration of the reaction mixture. These ions disrupt ionic bonds between DNA amd protein. Concentration of sodium acetate decreases the solublity of DNA in ethanol allowing precipitation.
ETHANOL – It is used to precipate DNA and remove it from the solution. Large DNA molecule can be seen with naked eyes and can be removed by a thin glass rod. Increased purification can be obtained using organic solvents like phenol and chloroform.
PRECAUTIONS : Care must be taken to avoid contamination by DNase. The enzymes can be under the glassware also. Latex gloves should be worn while isolating the DNA.
RESTRICTION DIGESTION OF λ DNA
AIM: To demonstrate the activity of restriction endonuclease digestion usinfg agarose gel electrophoresis.
PRINCIPLE:
The DNA from bacteriophage λ is the most widely used substrate for screening restriction enzymes because it is often difficult to determine a characteristics pattern of a λ digest.
The resulting DNA is displayed on a agarose gel and visualized by staining with ethidium bromide. A given sequence in DNA is recognized and can be cleaved by more than one restriction enzyme. The term isoschizoners describes a group of enzymes that recognize the same sequence in DNA.
Restriction endonucleus have their cleavage sites. The cleavage pattern of EcoRI is as follows. It produces cohesive ends, with 5 single stranded overhang. Under certain condition i.e. low ionic strength, alkaline pH or 50% glycerol, the EcoRI specificity is reduced.
REQUIREMENT :
(a) CHEMICALS REQUIRED :-I. Tris acetate EDTA (51X)
II. 2X Assay buffer – 25µlIII. EnzymeIV. Sample- λ DNA (25µl)V. Molecular weight marker-Hind III and EcoRI
VI. Gel loading dyeVII. EtBr-8µl
VIII. Agarose gel(1%)
(b)Role of Chemicals : -
I. TAE buffer (Tris acetate EDTA) – It is used as the running buffer.II. 2x Assay buffer – It is test buffer and its composition is tris-Hel.
III. EtBr (Ethidium Bromide) – it Provides fluorescene to the plasmid DNA under U.V
IV. Light, as Ethedium Bromide binds to DNA molecule by intecalating between adjacent base pair causing partial unwinding of the double helix.
V. Gel loadind dye - -It consist mainly bromo cresol blue or Bromo thymolVI. Blue or Bromo-phenolblue and glycerol – Bromo cresol or Bromo-thymol or
Bromo-phenol blue gives colour to the gel. These are called as indicator dye. These are more negatively charged than DNA, therefore it moves ahead of DNA when run in the gel electrophoresis. Glycerol is viscous. It gives density to the plasmic DNA to get settle in the well as the plasmic DNA is very light.
VII. Agarose gel – Greater the agarose conc. the smaller the pores, and lower the size range of DNA fragments that can be resolved.
MATERIALS REQUIRED :
Micropipetes, microtips, vials etc.
INSTRUMENTS REQUIRED:
a. Water bath b. Electrophoresis apparatusc. U.V transilluminator
PROCEDURE:
(a)The given enzyme and sample were kept in an ice bucket. (b) 25µl of λ DNA was added to the enzyme.(c)Equal quantity i.e. 25µl of buffer (2X Assay buffer) was added to the
vials.(d) The vials were gently tapped for a few seconds or briefly spun at low
speed in a microfuge.(e)After being tapped the vials were incubated at 37˚C in water bath for 1
hour.(f) As soon as incubation was over, adding arrested the reaction 10µl of gel
loading dye to the vials.(g) The sample was immediately run on the agarose gel for
electrophoresis.(h) The gel was electrophoresed at 50 V till the dye reaches about 3/4th the
length of the gel.(i) Visualized the gel under UV transilluminator.
OBSERVATION:
LANE:
C – Control λ DNA not digested
S1 – sample – 1
S2 – sample – 2
M1 – λ DNA digested with Hind III
M2 – λ DNA digested with EcoRI
DISCUSSION:
λ DNA is restricted by using restriction enzyme EcoRI. EcoRI recognizes the sequence 5’ – GAATTC. Since the cut is between G and A bases, this will lead to fragments having overlapping (sticky or cohesive ends).
Before Digestion After Digestion
5’ GAATTC 3’ 5’ – GAATTC – 3’
3’ CTTAAG 5’ 3’ – CTTAAG – 5’
Intermolecular Association –
5’AATT -------------------------
----------------------TTAA 5’ 5’ AATT -----------
----------
5’ AATT ------------------------ -------------------
TTAA 5’
---------------------- TTAA 5’
Base Base
Pairing Pairing
5’AATT ---------AATT ---------------
------------TTAA ------------TTAA 5’
From the observation, it is clear that C is controlled λ DNA of molecular weight, 48000 M1 is the molecular weight marker, which is being restricted by Hind III
Cohesive ends of DNA fragments produced by digestion of EcoRI
and M2 is the molecular weight marker, which is being restricted by EcoRI. It is seen that S2 fragments are coinciding with the M2 fragments, making it clear that the sample was restricted using EcoRI, as it is seen that S2 fragments are coinciding with the M2 fragments, it is clear that the sample was restricted using EcoRI and not by Hind III.
LIGATION
AIM:-
To ligate the digested DNA sample using DNA ligase enzyme.
INTRODUCTION:-
It may occur at random or because of DNA replication or recombination between 3’ –OH at the end of one chain and a 5’ –Po4 group at the other end. This process is DNA ligase is an important cellular enzyme as its function is to repair broken phosphodiester bonds that known as ligation. In genetic engineering, it is used to seal discontinuities in the sugar phosphate chains that arise when recombinant DNA is made by joining DNA molecules from different source. It can therefore be thought of as a molecule glue, which is used to stick pieces of DNA together. This function is to many experiments and DNA ligase is therefore a key enzyme in genetic engineering.
Invitro DNA ligation is performed by the action of DNA ligase that is E.coli ligase (NAD), T4 DNA ligase (ATP). The enzyme used most often in experiments is T4 DNA ligase which is purified from E.coli cells which are infected with T4 bacteriophage. Although the enzyme is most efficient when sealing gaps in fragments that are held together by cohesive ends, it will also join blunt ends of DNA molecules together under appropriate condition. The enzymes works best at 37 ˚c . Normally joining of two molecules by the action of ligase is with breakdown of pyrophosphate bond as the formation of the new phosphodiester bond requires. In case of E.coli, the action of ligase is to the cleavage of the pyrophosphate bond of the NAD+ while it is link to the cleavage of the α, β-pyrophosphate bond of ATP. The eukaryotic ligase also uses ATP.
E.coli DNA ligase consist of a single polypeptide chain of about 77,000 molecular weight. Each E.coli cell has about 200 to 400 copies of the DNA ligase. Fulfillment of the energy requirement during the reaction mechanism is unusual in the sence that NAD+, which is a coenzyme for the oxidation – reduction reactions, functions as a source of adenyle group whose main role in this reaction is in the energy transfer.
Several functions that have been assigned to DNA ligase are as follows:
(a) To cooperate with DNA polymerase in the replication of DNA(b) To repair single stranded nicks in the duplex DNA,(c) To join segments of DNA in the process of recombination which occurs during genetic transformation, transduction and lysogenation in bacteria and meiosis in eukaryotes,(d) To link the ends of the linear DNA duplexes to yield
PRINCIPLE:
We know that DNA ligase is an enzyme which can join the ends of two DNA chains by catalyzing the synthesis of a phosphodiester bond between 3’-OH and 5’-PO4 group. Ligation of one end of the DNA to another can be regarded as a biomolecular reaction whose velocity under standard condition is determined solely by the concentration of the compatible DNA termini. DNA ligase can form only single phosphodiester bond between the ends of the chains and it requires that the chains to be joined should be associated in double helix with a complimentary DNA strands and the residues which are joined be passed to adjacent bases in the other strand.
Here, the λ bacteriophage, T4 DNA ligase is the enzyme used because it will join the blunt ended DNA fragments efficiently under normal reaction conditions. The energy source for DNA ligase is ATP. It is used at much lower temperature to prevent thermal denaturation of the base paired region that hold the cohesive ends of the DNA molecule together and the anchorage annealing between the two segments of the DNA.
Assay buffer provides the medium for the ligation reaction to take place. This buffer contains ATP, MgCl2, tris EDTA. The ATP present in this is used by the T4 DNA ligase enzyme as a source of energy.
Gel loading dye, which contains sucrose, xylene cyanol and bromophenol blue is used to arrest the reaction. Sucrose makes the DNA heavy, xylene cyanol increases the density and bromophenol blue adds colour to the sample.
In the simplest case the construction of a recombinant plasmid is a biomolecular reaction in which the free ends of a linearise vector DNA are joined to be cloned. Therefore, one can adjust the conditions of a ligation reaction such as the formation of circular, linear or cloning of the desired DNA fragments.
REQUIRMENTS:-
(a) Glass wares – Beakers, conical flasks
(b) Instruments – Water bath, micro centrifuge
(c)Chemical reagents – Ligase assay buffer, gel loading dye
(d) Other requirements – λ DNA – EcoRI digested DNA sample, T4 DNA ligase, gel running buffer – TAE, agarose, ependroff tubes, micropipettes and micro tubes.
PROCEDURE:-
1) The given ligase assay buffer and the sample to be ligated was thawed completely and placed in ice.
2) 10 ml of given sample was added to the enzyme.3) 10 ml of 2x ligase assay buffer was added and mixed by tapping gently.4) The vial was incubated at 16 ˚c for about two hours and adding 5 ml of the
gel loading dye arrested the reaction.5) The sample was loaded in the gel and electrophore till the dye reaches half
the gel.6) Visualize the gel under UV light in a UV transilluminator.
OBSERVATION AND RESULT:
Ligation of λ DNA cleaved by EcoRI was done using the enzyme T4 ligase. After the end of the incubation period the sample was loaded on to an agarose gel and electrophorized. Under the transilluminator the gel appear as
Lane – 1: Control the well containing the sample with no T4 DNA ligase
Lane – 2: Shows partial ligation
Lane – 3: A single band of DNA is seen. Complete ligation
DISCUSSION:
λ DNA which was restricted by the enzyme EcoRI was used as the sample for ligation. T4 DNA ligase was the enzyme that was used. The ligase enzyme was added to the restricted sample along with the assay buffer and was incubated at 16 ˚c for 2 hours and this temperature is the optimum for the action of the ligase enzyme on the restricted DNA. After incubation period, the ligated sample was run on the agarose gel. These different samples were loaded along with one controlled sample (i.e.; the sample to which the ligase enzyme was not added) into the gel. The control was used for comparing the results obtained and the check the accuracy of the experiment.
The results obtained were as follows:
In lane 1, which was loaded with the control sample, showed two different bands which implies that the sample contains one restriction site because the ligase enzyme was not added to the sample, there were no action of the ligase enzyme and two discontinuous bands were seen, which was restricted by EcoRI confirming the presence of one restriction site.
In the remaining lanes, i.e. 2, 3,4,5,6,7and 8, single continuous bands were seen. In these lanes there was the action of the ligase enzyme and proper ligation of the λ DNA sample by the enzyme T4 DNA ligase has taken place.
Ligase catalyses the formation of phosphodiester bonds between the directly adjacent 3’-OH and 5’-PO4 termini of nucleic acid molecules. The substrate may be DNA or RNA and the cofactors that generate high energy intermediates in the reaction may be ATP or NAD+, depending upon the type of ligase.
Invitro DNA ligase is required for enzymatic completion of lagging strand synthesis during replication and DNA repair. Invitro DNA ligase is used chiefly to create novel combination of nucleic acid molecules and to attach them to vectors before molecular cloning. More specialized uses of DNA ligases include sealing
of nicks on the second strand during synthesis of complementary DNA, the amplification of segments outside the boundaries of known DNA sequences, the deletion of nicks in DNA by the release of ATP and more recently the deletion of point mutation in DNA by the ligase chain reaction.
The DNA ligase used in molecular cloning differs in their ability to ligate non substances, such as blunt ended duplexes DNA – RNA hybrids or single stranded DNA’s.
AGAROSE GEL ELECTROPHORESIS
AIM: To visualize the isolated plasmid DNA on the gel.
PRINCIPLE:
Gelation involves the transition of polymers from a random coil to a double helical conformation, the partners in a helix being different polymers or two segments of the same polymers. This produces a network of pores with diameter of 100-300 nm, the size range being dependent on a variety of factors, with the most important of these in practical terms being the agarose concentration. The greater the agarose concentration, the smaller pores and the lower the size range of DNA fragments that can be resolved. Gel concentration also affects the gelling and melting temperature and the gel strength, although the latter is also affected by temperature and (so low concentration gel is more easily handled in a cold room) the age of the agarose, since hydrolysis of the agarose, polymers occur overtime.
Agarose gel electrophoresis a simple and highly effective method for separation, identification and purification of DNA. This separation is carried out under an electric field applied to the gel matrix. DNA molecules migrate towards the anode due to negatively charged phosphates along the backbone of the DNA. The rate of migration of a linear DNA is inversely proportional to the ratio of its molecular weight. Thus the larger molecules travel at a much slower speed when compared to smaller ones. Several parameters like agarose concentration and the voltage applied affect the migration of DNA and these should be critically selected.
The DNA can be visualize using EtBr, which would intercalate between the base pairs of DNA in the gel and it would fluoresce under the presence of UV rays in an UV transilluminator.
REQUIRMENTS:
(a) Chemical Required:- 1. Agarose2. 1 X TAE Buffer3. EtBr4. Methanol
(b) Materials and Instruments:- 1. Electrophoresis Apparatus2. Cellophane tape3. Micropipette4. Transilluminator
PROCEDURE:
(a)300 ml of TAE buffer from a stock of 50X TAE was prepared.(b) 1% agarose in 50 ml of 1 X TAE was also prepared. It was then boiled to dissolve agarose.(c)The electrophoresis tank and cast was wiped with alcohol to make it grease free. Then cellophane tape was pasted on both the side of the cast to prevent the flow of liquid agarose.(d) The electrophoresis apparatus was then set.
(e)The gel was poured in the cast slowly without creating air bubbles and allowed to polymerize.(f) After polymerization the comb was removed and the cast transferred in the electrophoresis tank.(g) 1X TAE buffer was poured into the electrophoresis tank till the gel is submerged.(h) The gel was then ready for loading.(i) The sample was mixed with 5ul of gel loading dye and 25ul of the sample into the well.(j) The gel was electrophoresed at 50V till the dye reaches about 3/4th the length of the gel.(k) Then the gel was visualized under U.V in a transilluminator.
OBSERVATION:
Linear
Nicked
Supercoiled
DISCUSSION:
Control shows the presence of three bands i.e. the linear, nicked and super coiled form of DNA. The supercoiled form can travel the fastest due to its coiled nature followed by the nicked and the linear form.
Sample 3 & 4 show two bands, the lower one being the supercoiled DNA and the band formed above the supercoiled one is the nicked form of DNA. Linear form of DNA is not seen while all the other samples show only the presence of supercoiled DNA.
DNA ELUTION METHOD
AIM: To elute DNA from low melting temperature Agarose gels.
PRINCIPLE:
Agarose that has been modified by hydroxyethylation, a substitution that reduces the number of intrastrands hydrogen bonds, melts and sets at lower temperatures than standard agarose. These properties form the basis of techniques to recover and manipulate DNA fragments in gels. In addition to enzymatic reactions, low melting/gelling temperature, agarose may be used for rapid recovery of DNA from gels and for bacterial transformation with nucleic acids in the remelted gel.
Agarose gel should be free of DNAase and RNAase activity and to display minimal inhibition of restriction endonucleases and ligase.
DNA fragments are separated according to size by electrophoresis through melting temperature agarose. Located by staining with ethidium bromide and U.V light illumination and then recovered by melting the agarose and extracting with phenol : chloroform. The protocol works best for DNA fragments ranging in size from 0.5 kb to 5.0 kb.
MATERIALS REQUIRED:
BUFFER AND SOLUTION –
Ammonium acetate (10M)
Chloroform
Ethanol
Ethidium Bromide
6X gel loading buffer
LMT elution buffer
Phenol : chloroform (1:1)
Phenol, equilibrated to pH 8.0
1X TAE electrophoresis buffer
TE (pH 8.0
GEL –
Agarose gel made with low melting temperature agarose.
NUCLEIC ACID AND OLIGONUCLEOTIDES –
DNA sample.
PROCEDURE:
A few mins after electrophoresis, the position of the DNA is noted in the UV transilluminator.
An incision is made in the gel just ahead of the desired DNA band and the rectangular piece of the gel was taken out.
The space was cleaned and filled with 1 % low melting agarose with ethidium bromide and allowed to polymerize.
The gel was electrophoresed again till the DNA reached into the portion of the low melting agarose which could be monitored using the transilluminator or by noting the distance between the DNA and the gel loading dye in the gel.
This part of the gel was cut and put into a vial.
The low melting agarose was melted at 65 ˚c for 15 mins in a water bath. Equal volume of equiliberated phenol was added to the vial and boiled at
65˚c for one min. The vial is then centrifuge at 12000 rpm for 12 mins at room temperature. The supernatant is transferred into a fresh vial and equal volume of
phenol:chloroform:isoamyle mixture was added. It is then centrifuge at 12000 rpm for 12 mins at room temperature. The supernatant was transferred to a fresh vial and 0.1 volume of 3M
sodium-acetate and 2 volume of ice cold absolute alcohol are added and it is kept for precipitation overnight.
The pellet was washed with 70% ice cold ethanol. Centrifuge at 12000 rpm for 10 mins at 4 ˚c . The pellet is air dried and dissolved in 20µl of 1X TAE buffer.
RESULTS AND OBSERVATIONS:
The electrophoresed super coiled plasmid DNA from the gel is eluted out. On electrophoresis and visualization under the UV transilluminator the gel appeared as
Lane 1 – sample of plasmid DNA,directly isolated from E.coli.
Lane 2 – eluted DNA.
DISCUSSION:
The elution was done using low melting agarose, since the melting point of this much lower than the normal agarose. It enables one to recover the DNA without being denatured.
The cut and pour method was employed mainly due to economic reason, otherwise it would have been quite possible to initially use low melting agarose itself, this would not have made any differences.
The image of the agarose gel under the UV transilluminator can be explained as
Lane 1 – as in the previous experiment all the three forms of plasmid DNA is present.
Lane 2 – only supercoiled DNA that was eluted from the initial experiment. The band was much brighter than compared to lane 1 because the purity of the supercoiled plasmid DNA had increased.
ISOLATION OF TOTAL RNA FROM YEAST
AIM:
To isolate the total RNA from yeast cells.
PRINCIPLE:
Total yeast RNA is obtained by extracting a whole cell homogenate with phenol. The concentrated solution of phenol disrupts hydrogen bonding in macro molecules causing denaturation of protein, the turbid solution is centrifuged and two phases appears. The upper phenol phase which is aqueous contains DNA. Denatured protein which is present in both phases is removed by centrifugation. The RNA is precipitated by adding alcohol.
PROCEDURE:
(a)Suspend 5gm of dry yeast in 10ml of previously heated to 37 ˚c.(b) Incubate it at the same temperature for 15 mins and add 8ml of concentrated phenol solution.(c)Stir the solution mechanically for 30 mins at room temperature.(d) Add 1ml of the solution to a clean vial.(e) Centrifuge at 3000 rpm for 15 mins in the cold condition to break the emulsion.(f) Carefully remove the upper aqueous layer and centrifuge to separate the denatured protein. Take the supernatant and add 2 volumes of ice cold ethanol.(g) Incubate the vials in the refrigerator for 1 hour.(h) Collect the precipitate by centrifuging at 2000 rpm for 5mins in cold and decant the supernatant.
(i) Wash the RNA with ethanol : water (3:1) with 100ml by centrifuging at 2000 rpm for 2 mins and decant the supernatant.
(j) Wash the RNA with ether (100ml) by centrifuging at 2000 rpm for 2 mins and decant the supernatant.(k) Air dry the pellet in 200µl of TE buffer.(l) Load the agarose gel and visualize in UV transilluminator.
OBSERVATION:
The RNA was separated by centrifugation and treated with ether for purification. The precipitation was air dried overnight and later dissolved in TE buffer and observed by running the samples in the agarose gel.
RESULT:-
An orange band was observed at a similar position as that of the control RNA. The extracted RNA was seen ahead of the dye.
DISCUSSION:-
RNA is quite prone to degradation due to extremely resilient ribonucleases. RNase can survive even boiling temperatures hence, autoclaving is not entirely successful to remove it. To eliminate RNase all glasswares associated with RNA extractions should be baked at 180-200 ˚c for at least 4 hours. For removing RNase from solution the enzyme RNAasin can be used at 1000 units/ml.
Mini gels are run to check for the presence of nucleic acids. This is done for example, after an extraction protocol to determine whether DNA (or RNA) was successfully isolated or following a PCR amplification to determine whether the reactions were successful. Both DNA and RNA staying with ethidium bromide and will migrate to different positions based on their molecular weight.
The band of RNA on electrophoresis move faster than DNA hence, the band of RNA is observed ahead of tracking dye.
Plasmids are extra chromosomal genetic material and DNA. Various methods available for its isolation. Currently alkali lysis method has been employed to
isolate plasmids from E.coli. the plasmids thus extracted where visualized on an agarose gel. The samples were found to contain only supercoiled forms of DNA
along with RNA.
Agarose gel electrophoresis is an effective method separating the negatively charged DNA in the presence of electric current. DNA which is very small and invisible under normal condition to the naked eye can be visualize on the agarose gel under UV light.
This is very much essential in monitoring molecular biology experiments and rDNA technique which uses DNA extremely.
Gel elution is a process of separation of separated DNA into a buffer. The need for this technique arises when we have to get back the DNA from agarose gel. It is used to get back the gene into the buffer for further experiments. A highly purified forms of agarose called low melting agarose is used to get back the DNA undamaged.
Restriction digestion is a process of cleaving the double stranded DNA at specific recognition sites using specific restriction enzymes by breaking the phosphodiester bonds. The fragments thus obtained are predictable and hence are very useful in recombinant DNA technology to insert the gene of interest which initially requires cleavage of plasmids.
ɅDNA was used as the substrate and EcoRI was the enzyme. Cleavage occurred at 5 sites resulting in 6 fragments.
Ligation is a process of sealing single stranded DNA by formation of phosphodiester bonds. They are not site specific in their action. They are used in recombinant DNA technology to splice back the cleaved DNA after insertion of gene of intrest in the experiment, EcoRI digested ɅDNA was the substrate used and staggered and ligase was the enzyme used. The samples were effectively ligated back to a single fragment during the experiment.
DNA is the genetic material of all eukaryotic and most prokaryotes. It has to be from a cell for its analysis and various other experiments of the various methods available for genomic DNA extraction, chloroform method was employed in this experiment. The source used was plant leaves where in DNA was extracted progressively by first rupturing of cell wall and later by step wise elimination of unwanted components. Finally, DNA is precipitated and dissolved in a buffer for further purpose (here, for visualization on agarose).
RNA in the nucleic acid which assists in the functioning of the cell using DNA as a template. It is also the genetic material of a few prokaryotes. It has to be extracted out of the cell for its analysis and for various other reasons. Its extraction includes a detailed procedure in which the interfering components are eliminated at the end of each step. Finally, the RNA is precipitated out and dissolved in a buffer to store it until needed. The source used here is yeast cell.
1. Covalently closed circular DNA – The super coiled conformation can be maintained if both polynucleotide strand are intact, hence most technically it is maintained as CCC DNA2. Electrophoresis- Movement of charged particles under the influence of current.3. EtBr – Ethidium Bromide. It is carcinogenic. Under UV radiation it has the property to give fluorescence color to the DNA.4. Gel Loading Dye – it consists of mainly Bromo-Cresol blue or Bromo Thymol blue or Bromo Phenol blue and glycerol.5. Isochizomers – it describes a group of enzymes that recognize the same sequence in the DNA. 6. Module – it may be regarded as a DNA segment or sequence performing a specific function. Each module may contain one or more genes.7. OC – open circular: If one of the polynucleotide strands is broken the double helix reverts to a normal relaxed state and the plasmid takes on the alternative conformation called open circular.8. Palindromic sequence – in this, the base sequence in both the strands of a duplex reads the same when read from the same end of both the strands.9. Restriction sequence – the site recognized by the restriction endonucleases is called recognition sequence or recognition site.10. Spheroplast – bacterial cell with primarily wall cells that retain as intact cytoplasmic membrane.11. Unit of Restriction endonuclease activity – it is defined as the amount of enzyme required degrading 1 µg of DNA in 1 hour at 37˚C.
1. DNA loading dye:50% Glycerol0.25% Xylene Cyanol
2. 0.5M EDTA, pH 8.0:(a)Dissolve 93.05g in 300ml of water and 0.25% Bromophenol blue.(b) Adjust pH to 8.0 with conc. NaOH or NaOH pellets.(c)The EDTA will go into the solution as pH reaches 8.0.(d) Adjust the volume to 500ml in water.(e)Autoclave to sterilize.(f) Store at room temperature.
3. 10mg/ml ethidium bromide solution:(a)Dissolve in water.(b) Store at 4C at protected from light.
4. 10% SDS – sodium dodecuyle sulphate:(a)Dissolve 100gm of SDS in 80ml of warm water (60C)(b) Adjust the volume to 100ml.
5. Solution:50mM glucose.25mM Tris-HCL (pH-8.0)10mM EDTA (pH-8.0)
6. Solution – II:0.2N NaOH1% SDS.
7. Solution – III:3M sodium acetate or potassium acetate.Glacial acetic acid
8. Solution – IV:Ethanol 100%
9. TAE (Tris acetate EDTA):48.4 gm of Tris base.11.4ml of glacial acetic acid.7.44gm of N2 EDTA.2H2O
10. 1X TE:10mM Tris, pH 7.41mM EDTA, pH 8.0
11. Assay buffer:Tris – HCL
12. LB (Luria-betoni) broth (25ml)0.25gm of tryptone.0/125gm of yeast extract.0.25gm of NaCl.(a) Make 25ml by adding water. (b) Autocleave to sterilize.
B.D. Singh, biotechnology, Kalyani Publishers, New Delhi. Judith.A.Scheppler, Patricia.E.Cassin, Rosa.M.Gambier, Biotechnology
Exploration, ASM press, Washington DC. S.C.Rastogi, V.N.Sharma, Anuradha Tandon, Concept in Molecular
Biology, New Age International, Revised Edition, New Delhi. T.A.Brown, Gene cloning and DNA, Blackwell Science Limited Analysis,
4th Edition, Oxford USA. Jac.G.Chirikjian, Jones and Bartelett, Genetic Engineering, Mutagenesis
Separation Technology, London Publishers. Re.Fr Dr. S. Ignacimuthu, Methods in Biotechnology, New Delhi, Phoenix
Publishers. T.A.Brown, Molecular Biology-Gene Analysis, Academic Press, USA
Academic Press. T.A.Brown, Molecular Biology, LABFAX II Edition, Academic Press, USA
and I. Sambrook and Russell, Molecular Cloning, Cold Spring Harbor Lab,
Volume-I Third Edition, Press New York. Helen Krueger and Adrienne Massey, Recombinant DNA and Technology,
Second Edition, ASM pres, Washington DC. P.K.Gupta, Introduction to Biotechnology, Rastogy Publications, Merrut,
CBS publishers and Distributor, New Delhi. K.S.Bilgrani, A.K.Pandey, J.M.Walker and E.B.Gingold, Molecular Biology
and Biotechnology, Panima Educational Book Agency, New Delhi. R.W.Old and S.B.Primrose, Principles of Gene Manipulation, Blackwell
Science USA.