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Taxonomic Classification of Common Livestock Species

A series of terms (kingdom, phylum, class, order, family, genus, species etc) is used to denote the member of a kingdom and each division of the hierarchy contains one or more groups from the next lower division like subphyla, subclasses, suborders, subfamilies, or subgenera etc if required. Further, species may be subdivided into varieties, breeds, or strains. Domestic farm animals belong to either class Mammalia or Aves. The scientific name of an animal comprise of its genus and species names. The first letter of genus name is written in capital letter but not the species name. While writing the scientific name of an animal the genus and species names are underlined when handwritten and always italicized when printed. General characteristics and taxonomic arrangements of common domestic animals have been given below.

Kingdom : Animalia

Members of kingdom Animalia are multicellular, eukaryotic heterotrophs. Adult animals develop from embryos. Most animals ingest and digest their food in some kind of internal cavity.

Administration

Term Administration is derived from a Latin word Administaire which means to serve. The word administration is also derived from the English word administracioun, which is in turn is derived from the French word administration, which itself has its root from the Latin word administratio, which means ad (to) and ministratio (give service). The term administration is a more general and comprehensive connotation and may be defined as the universal process of organizing people and resources efficiently so as to direct activities toward common goals and objectives. Administration is a distinct skill and directed towards the guidance, leadership and control of the efforts of a group of individuals towards some common goal.

Administration is the art of guiding, coordinating and integrating policies established for any organizational unit to effectively and efficiently achieve the purpose of the organization.

Nearly 50 percent of the world’s population lives in urban areas, it is not possible that food production be adequate for their needs. In some areas of food production surplus, it must be brought to the deficit places where peoples live and work and made available at prices they can afford, otherwise many will remain hungry. This is why marketing is a crucial part of any campaign to relieve hunger. Marketing is the means by which food is brought from the place where it is produced to the people who want to eat it.

Marketing can be a very simple process and it can also be very complex. This depends on what must be done to the farmer’s product to get it from the field to the consumer in good conditions, in an acceptable form and to have it ready when the consumer wants.

Marketing is relatively simple in a village community which produces most of what it needs. Here the producer can sell directly to the consumers coming to his house, or he can attract them by displaying his produce in some central open space in the village which then becomes known as ‘the market’. The total volume of the livestock products sold in this way is still very large, especially in the less commercially developed countries. Direct marketing within the villages is sometimes praised because of its low cost, and also because it does not involve any ‘middleman’ between producer and consumer. But, if farmers sell their produce only to the local consumers, and if their land could produce more than they need, there is no way by which they can take advantage of these excess production potentialities. Similarly, if the consumers are only able to eat what is produced locally, which may confine them to a diet that is monotonous, unsatisfying and very expensive too. This is true of many people’s living in isolated areas, which requires much home processing and still has little nutritional value; and of the many others for whom most meals consist of hand-pounded cereals. Hence, this undernourishment on one side and excess production on the other side leads to the development of marketing concept.

Farming system is an integrated set of activities that farmers perform in their farms with available resources and circumstances to maximize the productivity and net farm income on a sustainable basis. The farming system conceptually is a set of elements or components, i.e., soil, water, crops, livestock, labour, capital, energy and other resources that are interrelated and interact among themselves. At the centre of the interaction lies the farmer exercising control and choice regarding the types of results of interaction. The income of cropping alone from small and marginal farm is insufficient now to sustain the farmers family. A judicious mix of any one or more of these enterprises should complement the farm income and help in recycling the farm residues / wastes. The selection of enterprises must be based on the cardinal principles of minimizing the competition and maximizing the complementary between the enterprises. Of late, the researchers on multidisciplinary approach greatly realized and started developing the various farming systems models in accordance with the agro-eco systems zones.

Farming System thus may be defined as a complex interrelated matrix of soil, plants, animals, implements, power, labour, capital and other inputs controlled in part by farming families and influenced to varying degrees by political, economic, institutional and social forces that operate at many levels. The farming system therefore, refers to the farm as an entity of inter-dependent farming enterprises carried out on the farm. The farm is viewed in a holistic manner and the farmers are subjected to many socio-economic, biophysical, institutional, administrative and technological constraints.

Rural development programmes are created and maintained for influencing people to make changes in their way of living and of making a living. The existence of such programmes implies that the present situation of the rural people is not what it should and could be, that something different should prevail, and that it is possible through appropriate action to attain a more desirable status for them.

Changes that are important to people are those that help them to meet their needs for biological, economic, social, aesthetic and moral well being. Basically, all programmes for rural development exist to help people to meet their needs. If programmes do this people have their free choices in societies and will not participate for long periods in them. The element of people’s needs, therefore, becomes a central concern of planner for rural development. Hence, the entire process of planning for rural development implies a need for change. But now the question arises that change from what, to what, by whom, where and by what methods?

The society has to find ways to adjust itself so that people meet their needs for an improved economic status, for being more useful citizens and for enjoying a higher degree of social stratification, it must identify these conditions and plan realistically to achieve them. It is the prime concern to identify that what needs do people have ? How can needs be identified? What plan of priority should be set up for meeting people’s needs? What resources are necessary to meet people’s needs? How should resources be organized and directed to help people’s meet their needs? These questions give the key orientation and justification for rural programme planning.

One of the major constraints in the efforts to modernize and efficient restructuring of the animal husbandry practices is the delay in taking the results of research to the farming community and in persuading them to adopt new, profitable and locally relevant technologies. The very need of the extension work is to bridge the gap between what is known to the scientists in the laboratories and what is actually being practiced by the farmers in the field. In other words, the major goal of extension machinery is to accelerate the processes of communication and to motivate rural people or livestock owners for adoption of new and profitable technologies. Hence, it is very important to understand these processes of how the farmers get such technologies and how they adopt them.

Communication

The term communication is originated from the Latin word ‘Communis’, meaning common or establishing commoness. When we communicate, we attempt to establish ‘commonality’ with someone through the message.

Communication is the process by which two or more people exchange ideas, facts, feelings or impressions in ways that each gains a common understanding of the meaning, intent and use of the message (Paul Leagens, 1961).

The basic aim of extension system is to bring about a change in the behaviour of the people with whom the work is done. Change in behaviour pertains to change in knowledge, skills, attitude, understanding, etc about a particular technology which we want to diffuse. For this purpose, the extension workers essentially function as teachers to bring about adoption of new technologies by making regular contact with livestock owners through various means. He visualizes the problems of rural community in proper perspective and organizes the meaningful learning situation for effective learning. It calls for appropriate use of different extension teaching techniques. The desirable changes in the behaviour of learners depend upon the skill of the extension worker as teacher in choosing the various extension teaching techniques which are found to be most effective in the relevant situations.

The main job of the extension worker is to bring about behavioural changes in the rural people. A number of extension methods are required to perform such behavioural changes among the rural people which form the tools of the extension workers. These tools are called extension teaching methods. Extension teaching methods may be defined as the devices used to create situations in which communication can take place between the extension worker and learner. The effectiveness of various teaching methods depends on the combination of the same. Similarly, the adoption of innovation is positively related to the combination of different extension teaching methods, at different stages of the learning process.

Audio-visual aids may be defined as any device used to aid in the communication of an idea. An audiovisual product is any audiovisual item such as still photography, motion picture, audio or video tape, slide or film strip, that is prepared singly or in combination to communicate information or to elicit a desired audience response. Even though early aids such as maps and drawings are still in use, advances in the audiovisual field have opened up new methods of presenting these aids, such as videotapes and other equipments which allow more professional and entertaining presentations to be presented.

Such aids assist the teacher in communicating information among the learners leading to change in skills, attitudes, knowledge, understanding and appreciation. These aids stimulates the sensory organs like ears, eyes, etc and facilitates quick understanding of the message by the audience. The effective use of these aids depends upon the skillful and imaginative approach of the teacher. Hence, it is necessary that extension worker should know what type of aids will be more suitable for a particular teaching objective. These aids may be used for both literate as well as illiterate people.

The instructional devices through which the messages can only be heard are known as Audio Aids. The instructional devices through which the message can only be seen are known as Visual Aids. The instructional devices through which the message can both be heard and seen simultaneously are known as audio-visual aids.

Cooperative

A cooperative is a duly registered association of persons, with a common bond of interest that have voluntarily joined together to achieve a lawful common social or economic end, and making equitable contributions to the capital required and accepting a fair share of the risks and benefits of the undertaking in accordance with universally accepted cooperative principles.

Cooperative may be defined as an autonomous association of persons united voluntarily to meet their common economic, social, and cultural needs and aspirations through a jointly-owned and democratically-controlled enterprise. It is a business organization owned and operated by a group of individuals for their mutual benefit.

A cooperative may also be defined as a business owned & controlled equally by the people who use its services or who work at it.

The basic objective of every cooperative is to provide goods and services to its members and thus enable them to attain increased income and savings, investments, productivity and purchasing power and promote among them equitable distribution of net surplus through maximum utilization of economies of scale, cost-sharing and risk-sharing without however, conducting the affairs of the cooperative for missionary or charitable purposes.

The livestock sector in India is experiencing fast growth in the present era and plays an important role in the Indian economy. Livestock contributes nearly 25% to the agricultural sector output and growing at an annual rate of 4.3% higher than the growth in agricultural sector as a whole (2.8%). Milk production that had been hovering around 20 million tones in 1960’s increased to 102 million tonnes in 2007-08. The egg production also increased from 0.1 billion to 1.0 billion in the last 25 years. Besides production of milk, meat, etc livestock make important contributions to crop production by supplying draught power and dung manure.

Animal husbandry in India is largely a rural activity. Rapid growth in livestock production is desirable not only to sustain agricultural growth, but also to reduce rural poverty especially when a majority of the land holdings are small. Fifty-eight percent of rural households have land holding of less than 2 ha and another thirty two percent have no access to land. Number of these households is likely to increase due to further fragmentation of land holdings. Livestock are thus an important source of income for small farmers, marginal farmers and landless agricultural labourers in the rural areas of the country. Products like milk and eggs are steady source of cash income, and live animals are important natural assets for the poor, which can be easily liquidated for cash during emergency. Smallholders and landless together control 75% of the country’s livestock resources, and are capable of producing at a lower cost because of availability of sufficient labour with them. These smallholders earn nearly half of their income from livestock sector.

India has been a welfare country since Independence and the primary objective of all governmental and non-governmental endeavors has been the welfare of its millions people. The policies and programmes should be designed with the aim of alleviation of rural poverty which has been one of the primary objectives of planned development in India. It was realized from pre-independent rural development efforts, that a sustainable strategy of poverty alleviation must be based on increasing the productive employment opportunities in the process of growth itself. Elimination of poverty, ignorance, diseases and inequality of opportunities and providing a better and higher quality of life were the basic premises upon which all the plans and blue-prints of development should built.

Rural development implies both the economic betterment of people as well as greater social transformation. In order to provide the rural people with better prospects for economic development, increased participation of people in the rural development programmes, decentralization of planning, better enforcement of land reforms, greater access to credit, etc proper planning is envisaged.

Initially, main thrust for development was laid on agriculture industry, communication, education, health and allied sectors but later on it was realized that accelerated development can be achieved only if governmental efforts are adequately supplemented with direct and indirect involvement of people at the grass root level.

Introduction

Livestock sector plays a significant role in the Indian economy, particularly for the welfare of rural population of India. The importance of livestock in India's economy can be gauged from the fact that 90 million farming families rear over 90 million milch animals. Of the total household in the rural areas, about 73 percent own some form of livestock, three-quarter of which are reared by small and marginal farmers.

Today, the overall growth rate in livestock sector is steady and is around 4-5%, despite the fact that investment in this sector is not substantial. This is possible due to the egalitarian distribution of the livestock as compared to land among landless labourers, small and marginal farmers thus resulting in balanced development of the rural economy particularly in reducing the poverty especially among the weaker sections of the society. Livestock also plays an important source of employment in rural India, especially for women. The rural woman plays a significant role in most of the animal husbandry operations related to feeding, breeding, management and healthcare of the livestock.

Livestock plays an important and vital role in providing nutritive food to families both in rural and urban areas. Since the dawn of civilization, mankind has been utilizing different animal species for a variety of purposes, viz., production of milk, meat, wool, egg and leather, draught power, companionship, entertainment, research experimentation, sports, security, etc. Livestock wealth is deemed to be as the oldest wealth resource for mankind and was once a symbol of economic status in the society. This is one sector where poor contributes to the growth directly instead of getting benefit from growth generated elsewhere. The effect of livestock sector growth on poverty is anecdotal, yet scattered evidence on livestock's contribution to income indicates that growth in livestock production is pro-poor.

“It is unbelievable that such a tiny streamlet, as was our initial test trial with the first bacterial starters exactly 40 years ago, grew to a big river for an important industry. Who would have believed then that the use of starter cultures would be a matter of course today” (Niinivaara, 1994). The idea of inoculating Lactobacillus into dry sausage material was introduced by Jensen and Paddock in 1940(US Patent 2,225,783) with the aim to reduce the ripening time as well as ensuring the quality and aroma of dry sausages. The first LAB meat starter culture, introduced as a pure culture of Pediococcus cerevisiae, was developed in 1955 in the USA by Niven et al. (1955). At the same time, Niinivaara (1955) applied Micrococcus M53 to dry sausage production in Europe. The work of Niinivaara was continued byNurmi (1966) who combined micrococci with Lactobacillus plantarum. Today, several companies provide Lactobacillus sp., Pediococcus acidilactici, P. pentosaceus, Staphylococcus xylosus or S. carnosus strains in pure cultures or their mixtures for fermentation of meat (Hammes et al. 1985). A starter culture may be defined as a preparation or material containing large numbers of variable microorganisms, which may be added to accelerate a fermentation process. Being adapted to the substrate, a typical starter facilitates improved control of a fermentation process and predictability of its products (Holzapfel, 1997).

In 1908, Metchnikoff, the father of modern Immunology, wrote “Prolongation of Life” and created the incredible ongoing revolution known as the “Probiotic Revolution”. He proposed that “Lactic Acid Bacteria” (LAB) could render a great service in the fight against intestinal putrefaction and might postpone and ameliorate old age. Till then today, some 95 years later, dozens of the worlds most brilliant minds carry on the astonishing work that has scientifically been proven to generate restorative and healing effects in the body.

In the late 19^th century, microbiologists identified microflora in the gastrointestinal (GI) tracts of healthy individuals that differed from those found in diseased individuals. These beneficial microflora found in the GI tract were termed probiotics. The term ‘Probiotics’ is originally a Greek word meaning ‘for life’. Parker (1974) for the first time used this word. He defined Probiotics as “organisms and substances, which contribute to intestinal microbial balance”. But the most accepted definition of Probiotics was given byFuller (1989). He redefined Probiotics as “a live microbial feed supplement, which beneficially affects the host animal by improving its microbial balance”. Fuller’s definition since been broaden to state “a probiotic is a mono or mixed culture of live microorganisms which, when applied to animal or man, affect the host beneficially by improving the properties of the indigenous microflora”. This definition stresses the importance of live microorganisms that improve the health status of either man or animal and that occur in the mouth, gastrointestinal tracts (GIT), or upper respiratory or urogenital tracts (Havenaar et al, 1992).

To understand how probiotics work, it is important to understand a little about the physiology, microbiology of GI tract and the digestive process. The digestive process begins as soon as food enters the mouth and to stomach, the microbes present in the GI tract have the potential to act in a favourable, a deleterious or a natural manner. Microbes in small intestine and in large intestine complete the digestion process.

India is blessed with a coast line of 8118 km with an Exclusive Economic Zone (EEZ) of 2.02 million square km and a continental shelf of 0.506 million sq. km. The inland water resources includes 191024 km of rivers and canals; 2.05 million ha of reservoirs; 2.254 million ha of ponds and tanks; 1.3 million ha of oxbow lakes and derelict waters; and 1.24 million ha brackish waters. India has an estimated fish production potential of 8.4 million tons, of which the marine sector forms 3.9 million tons and the inland sector 4.5 million tons. As against this, the current fish production is 5.96 million tons i.e., 2.89 million tons from marine sector and 3.07 million tons from inland sector (Vannuccini, 2003). The per capita fish availability in India is 4.7 kg/year (Laurenti, 2002).

The traditional fermentation process is an uncontrolled system and is usually carried out in a quite unhygienic environment. As the process of fermentation is brought about by the microbial activity so in the traditional system of fermentation there is every possibility of inclusion of microorganisms which is not desired in or that can cause spoilage to the finished product. Although no incidents of food poisoning has been reported due to consumption of fermented fish there are also no reports of any clinical investigations in this regard. A study on the microflora of such a fermented fish product is useful to understand -how safe is the product for consumption. The diverse groups of microorganisms that are present in the fermented fish products will provide information about their characteristic nature -beneficial or pathogenic, their contribution towards production of flavour and aroma, etc. The environment, however, is the key factor for the so called ‘microbial diversity’ among different fermented fish products round the globe. Moreover, the aquatic habitat, processing technique and of course, the handling during processing are the other factors that equally make a way for the introduction of a variety of microorganisms in the finished product. A study on different curing stages i.e. fresh, semi-cured and fermented, is therefore necessary to understand the pattern of microbes and become evident the role played by the microbes during fermentation.

It is an old practice that during the flourishing period, the fishes specially trash fishes are caught from their natural habitat and a bulk of them are preserved to make available for further use. Traditional fish preservation techniques are mainly based on combination of salting, drying and smoking. These techniques have allowed more widespread consumption of fish. Sun drying is most ancient method of fish preservation. Drying removes moisture from tissues and help to arrest bacterial and enzymatic putrefaction.

Fermented fish have, for many years, been considered as a Southeast Asian product. These products are highly salted and fermented until the flesh is transformed into simpler components. Fermentation of fish is a product in which the identity of the fish is maintained to large extent. Fermented fish products as fish sauces, fish paste, or salted fish have been consumed since ancient times. Because of poor roads and other methods of transport, the provision of fresh fish to potential inland consumers was impossible, and this encouraged fermentation as a preservation technique. In Southeast Asian countries such as Thailand, Kampuchea, Malaysia, the Philippines, and Indonesia, the use of fermentation as a preservation method for fish has been of great value since earliest times. In the countries of northern Europe, fermented fish products are used mainly as condiments, whereas in Southeast Asia, various fish products are regarded as staples. In India and Sri Lanka, pickled and in Colombo, cured fish have been known for many years

Fermented foods are food substrates that are produced due to the cumulative activity, usually of edible microorganisms, whose enzymes, particularly amylases, proteases and lipases hydrolyze the polysaccharides, proteins and lipids to non-toxic products with flavours, aromas and textures pleasant and attractive to the human consumer. The role played by fermentation during food processing helps in – enrichment of human dietary through development of wide diversity of flavours, aromas and textures in food; preservation of substantial amounts of food through lactic acid, alcoholic, acetic acid, alkaline fermentation and high salt fermentations; enrichment of food substrates biologically with vitamins, protein, essential amino acids, essential fatty acids and antibiotics; detoxification during food fermentation processing and reducing the cooking times and fuel requirements.

After preparing the desired DNA molecule using E.coli, the plasmid can be transferred into Agrobacterium by this method. Efficiency is low (~10³ transformants per mg of DNA) but the method is reliable and very rapid. This method also eliminates much of the plasmid rearrangement that occurs during triparental mating.

Procedure

Grow an Agribacterium strain containing an appropriate helper Ti plasmid in 5 ml of LB medium 16 hours at 28ºC.

Add 2 ml of the culture to 50 ml LB medium in a 250 ml flask and shake (~250 RPM) at 28ºC until an OD₆₀₀ of 0.5 to 1.0 is obtained.

Chill the culture on ice. Centrifuge the cell suspension at 1000 g for 30 minutes at 4ºC.

Discard the supernatant. Resuspend the cells in 1 ml of 20 mM CaCl₂ (ice cold). Dispense 100 mL aliquots into pre-chilled 1.5 ml tubes.

Add about 1 µg of the desired plasmid DNA (pure or crude) to the cells in each tube.

This protocol is designed for potato stem internodes on ZIG medium, but might work for leaf pieces as well.

Plant Material

Cut stem internode explants from 5-6-week-old in vitro plants (or leaf explants from 3-4-week-old in vitro plants) grown on 15 ml PROP in 25 x 150 mm test tubes.

Day 1

Streak out desired Agrobacterium strain on YEP + antibiotic to obtain a single colony. Grow at 28 C for 2 days. Alternatively, skip this step and use frozen stock for next step.

Day 3

Use a single Agrobacterium colony to inoculate 20 ml YEP + antibiotic in 250-ml flask. Grow overnight at 28 C with vigorous shaking (~200 rpm). Some Agrobacterium strains require an additional day to reach sufficient density.

Principle

Micropropagation is especially useful for include propagation of high value plants that grow slowly (bulbs, ferns, orchids, palms, etc), new cultivars having limited stock and high market demand (new cultivars produced via conventional breeding or genetic engineering), cultivars that are difficult to root by conventional techniques, and endangered plant species. In addition, virus elimination is possible via culture initiation from meristem explants. Also, micropropagation provides a source of sterile tissues that can be used for other tissue culture protocols, including genetic transformation.

Micropropagation is possible using multiple types of cultures; however, shoot cultures are typically employed, in which case five stages to the micropropagation process are typically recognized as outlined below. Nodal explants of rose are used for culture initiation. Three media, differing in hormone content, is employed and the explants should be evaluated for differences in axillary shoot growth and proliferation.

Principle

Orchid seeds are produced in large numbers but are exceedingly small, hence are also called “dust seeds” may be as small as 0.01 by 0.05 mm in dimension and weigh as little as 10-30 ug. Seedling development is generally a slow process in orchids, with the seedlings passing through an initial stage, termed the protocorm stage, prior to the production of distinct vegetative organs (leaves, roots, etc). The protocorms of terrestrial orchids are subterranean and entirely heterotrophic, with all of the nutritional needs of the orchid being supplied by the mycorrhizae (the symbiotic fungus). The length of the protocorm stage is variable, however, in many cases, months, or even years, elapse before leaves and roots are produced.

The combination of a small seed size and fungal symbiont requirement largely precludes the propagation via conventional seed propagation protocols. Accordingly, most of the early research on the development of orchid seed germination protocols focused on in vitro germination with the seeds and fungal symbiont being sown on the same petri plate. Successful protocols were developed for several species; however, the process was complicated by the diversity of fungal symbionts required (across orchid taxa) and the difficulty in isolating and culturing these fungi.

In the 1920’s the process of orchid seed propagation was revolutionized by Lewis Knudson with the demonstration that nutrient media that could be used as a substitute for fungal infection. The isolation and culture of multiple fungal strains was no longer required, and improvements in germination frequency, germination speed, and seedling vigor were generally observed. To date, successful asymbiotic germination techniques, termed ‘flasking’, have been developed for many epiphytic orchids, with several species being saved from extinction via the application of these techniques.

Media Preparation

Before mixing medium, determine the quantity of each medium and the amount of plant growth regulator needed for each experiment. Make slightly more medium than is required. A media check off list is helpful in making medium to keep track of ingredients.

Remove media stock solutions and plant growth regulator stock solutions to be used from the refrigerator. Make up fresh stock solutions if adequate quantities are not available or if solutions have precipitated.

Plant growth regulators stock solutions are prepared biweekly and stored at 4º C in the dark. Cytokinins (thidiazuron and BA) are dissolved in approximately KOH prior the addition of deionized water. Gibberellins and auxins (2,4-D, IBA and NAA) are dissolved in 100% ethanol prior the addition of water.

Fill the volumetric flasks upto ½ to ¾ with deionized water. Add proper a of stock solution and plant growth regulators and mark each item on the check off list. Add 30 g/l sucrose, stir until dissolved. Make up the volume of the flask with deionized water and later por into a larger beaker.

Principle

Western blot analysis (also called protein immunoblot) is a useful tool for the detection of proteins in biological samples. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose), where they are probed (detected) using antibodies specific to the target protein.  It is a technique for analyzing and identifying protein antigens: the proteins are separated by electrophoresis in polyacrylamide gel, then transferred (“blotted”) onto a nitrocellulose membrane or treated paper, where they bind in the same pattern as they formed in the gel. The antigen is overlaid first with antibody, then with anti-immunoglobulin or protein A labeled with radioisotope, fluorescent dye, or enzyme. 

The method originated from the laboratory of George Stark at Stanford. The name “Western blot” was given to the technique by W. Neal Burnette and is a play on the name “Southern blot”, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting and the detection of posttranslational modification of protein is termed “Eastern blotting”.

Cell Lysis Procedure

Pellet the cells in a microfuge tube: add 1.5 ml culture, centrifuge for 15 seconds at maximum speed, discard the supernatant, and then repeat the procedure. This will result in the cells from 3 ml of culture in a single tube.

Resuspend the cells in 1 ml Lysis buffer.

Take 300 µl aliquot for Bradford protein assay.

Add 50 µl lysozyme, and mix gently.

After 10-20 minutes at room temperature, microfuge for 10 minutes to pellet cell debris.

Use supernatant for LDH assay.

Principle

The SDS polyacrylamide gel electrophoresis (PAGE) performs:

Separation of proteins according to their molecular weight

Ascertain whether a given protein is actually present in a sample,

Assessment of purity of the preparation

Estimation of the approximate quantity of the protein, and

Measurement of the size of the protein.

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

The principle of silver nitrate staining of proteins is based on the reduction of silver ions to its metallic form. (Silver ions in alkaline conditions bind with proteins through å-amino group of lysine and sulphur groups of cysteine and methionine residues). When the complexed silver ions are reduced in the presence of formaldehyde, they become metallic silver, which is seen on the gel as bands. Staining of proteins with silver nitrate after SDS-PAGE is quicker and ~ 10 times more sensitive than Coomassie brilliant blue staining and that is the reason it is widely used in research laboratories these days.

The procedure is as follows:

Denaturing polyacrylamide/urea gels in TBE buffer

Prepare 20ml of a 5% polyacrylamide gel containing 7M urea by adding:

47.5% acrylamide:

2.5% bis-acrylamide solution 2ml

10M urea 14ml

10X TBE buffer 2ml

10% freshly prepared ammonium persulfate 0.2ml

deionized water 1.8ml

Principle

Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N’N’N’N’-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration: usually between 3.5 and 20%. Cross-linking bisacrylamide is usually added at a ratio of 2g BIS 38g acrylamide.

Principle

RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode. RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form. The following protocol is based on Formaldehyde Agarose Gel Method.

Principle

To retrieve and purify any specific DNA fragment from an agarose gel slice; the expected yield is from 50-75% of the amount in the gel slice.

Time required

Restriction digest - minimum of 2 hours

Electrophoresis - approx. 4 hours

Elution - 2-5 hours depending on fragment size

DNA Purification -2 hours

here are many methods for extracting DNA from gel apart from using commercial kits, some methods however yield cleaner DNA than others.

1. Freeze squeeze

Excise band and place pieces of it in a home-made spin-column (a piece of filter placed in a PCR 0.5ml eppendorf, pierced the bottom of the PCR tube with a needle). Place the PCR tube at -20 °C for 15-20 mins or freeze in liquid N2. Then place the PCR tube into the 1.5 ml eppendorf and spin at top speed in a microfuge for 15 mins, collect DNA solution which can be EtOH ppt or use directly.

2. Powdered silica

Melt excised bands at 70°C cool and add equal volume of TE-buffered phenol and mix. Spin for 5 min, remove aqueous phase, repeat and EtOh ppt. the DNA.

3. Electroelution

Agarose gel electrophoresis is the easiest and commonest way of separating, capable of resolving mixtures of DNA fragment that cannot be separated conveniently by other procedures. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualised in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light.

Agarose, which is extracted from seaweed, is a linear polymer whose basic structure is shown in figure.

Principle

The principle of Electrophoresis involves the motion of a particle in a fluid medium, where the gravitational attraction (on a flowing liquid) is the sedimenting force, and the frictional force is the opposing force and is proportional to the velocity of the particle. These forces balance each other acting in opposite directions and hence balance each other. This will lead to the uniform motion of the particle in the mobile liquid medium (i.e. the particle moves at constant velocity). In case the external field is an electric field instead of a gravitational field, a macromolecule will respond to the external field in two ways. In case the molecule is charged, it will migrate in an electric field to the electrode of opposite charge. Under this principle the technique of electrophoresis functions.

All electrophoresis operations are governed by the principle:

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

Transformation

The uptake of DNA by competent cells is called transformation. Transformation involves:

Mix a small volume of DNA (usually 1-3 µl) with 100 µl of competent cells.

The mixture is incubated on ice for 30 minutes to allow the cells to take up the DNA.

The cells are then heat-shocked at 37 to 42°C for 1 minute, and cold shocked on ice for 2 minutes.

The heat shock/cold shock reverses the effect of the competent cell process, by healing the damage inflicted by the competent cell solution.

Add 0.5 ml of LB culture mediumz

Incubate the cells for 10 - 30 minutes at 37°C to aid the healing process.

This cell/medium mixture is then spread onto bacterial culture plates containing agar, LB, and a selection mechanism.

Steps for Cloning

Prepare DNA.

Select restriction enzymes for your insert and vector, and determine the appropriate reaction buffers.

Combine the following in a microfuge tube (30 µL total volume):
2 µg DNA
1 µL Each Restriction Enzyme
3 µL 10x Buffer
3 µL 10x BSA
x µL H₂O (to bring total volume to 30 µL)

Principle

Plasmids requires hosts to replicate, hence it has to be inserted into bacteria. But efficiency of uptake of plasmid DNA by bacteria is low and will not do so without getting degraded. Therefore, to enhance the probability of uptake of the plasmid DNA by bacteria it is necessary to make the bacterial cells “competent” to absorb the plasmid DNA. One of the methods to increase efficiency of cell competence is given below. It should be noted that competent cells are more fragile than normal bacteria. Vortexing the cells, heating the cells above 42ºC or to 42°C for prolonged periods, or exposure of the cells to abusive treatments may kill them

Principle

The rate of joining of DNA fragments is affected by seven parameters:

DNA concentration,

Concentration of compatible ends,

Size of the fragments,

Presence or absence of complementary, single-stranded ends (some nucleases form blunt ends and in these cases special techniques are needed for joining),

G + C content and length of the complementary single strands,

Temperature of the reaction and

Ionic composition of the solution.

Start with a 1:2 molar ratio of vector: insert DNA when cloning a fragment into a plasmid vector. This ratio will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

Principle of DNA Ligase Mechanism

The process of connecting two pieces of DNA together is called ligation, and is catalyzed by an enzyme called ligase. The ligase used most often in molecular biology is derived from the T4 bacteriophage, and uses ATP to supply the energy necessary for the reaction. In addition to ATP, T4 ligase requires DNA with a 5´-phosphate group and free 3´-hydroxyl groups.

Applications

Ligation of blunt or cohesive-ended DNA fragments.

Repair of nicks in double-stranded nucleic acids.

Reagents

Restriction enzymes of choice, such as BamH1 and EcoRI

Restriction enzyme reaction buffer,

70 % Ethanol

100 % Ethanol

3 M Sodium acetate (pH 5.2)

Distilled water

Plasmid DNA isolated from bacteria, such as pGFP(R) and pBC(R)

Reagents

Nuclease-Free Water: 14µl

10X Restriction Buffer: 2µl

Acetylated BSA (1mg/ml): 2µl

DNA (~1µg): 1µl

Restriction Enzyme (10u): 1µl

Final Volume: 20µl

Principle

Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated. This restriction enzyme digest will create a pattern of specific DNA fragments that will serves as a “DNA ladder”.

Principle

Restriction digestion is one of the most common reactions performed in molecular biology. Restriction endonucleases (RE) are also known as restriction enzymes, which recognize short, specific DNA sequences. There are three types of RE: Type-I, Type-II and Type-III. Type II RE, most commonly used for DNA analysis and genetic engineering, cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. All Type II RE recognises a unique nucleotide sequence at which it cuts a DNA molecule and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences. Many RE will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation.

REs can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or “blunt end”. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called “cohesive ends”, as these single-stranded overhangs could potentially come together again through complementary base-pairing, as shown below:

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Is amyl alcohol

Phenol

Proteinase K

RNase A

Sodium acetate, pH 5.2

Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M

TE Buffer (Tris-EDTA), pH 7.4

Tween 20

Xylene

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Isoamyl alcohol

Phenol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

RNAse A

Sodium dodecyl sulfate (SDS) solution, 10%

Reagents

EDTA

Ethanol, absolute

Isopropanol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

Sodium Acetate, 3 M pH 5.2 (Molecular Biology Grade)

Sodium chloride (NaCl)

Sodium Dodecyl Sulfate (SDS ), 10%

Tris EDTA (TE), pH 7.4

Tris EDTA (TE), pH 8.0

Tris HCl, pH 8.0

Transfer 1.5 mL overnight culture to a 1.5 mL microfuge tube, centrifuge for 30 sec, and decant supernatant.

Resuspend cells in 400 µL TE by vortexing, add 50 µL 10% SDS, 50 µL proteinase K (20 mg/mL in TE). Incubate for 1 hour at 37^oC.

Shear DNA by 3-5 passages through a 26 G needle.
Extract twice with 500 µL phenol: chloroform (1:1), and twice with 500 µL chloroform. 
Precipitate nucleic acids by adding 25 µL 5 M NaCl and 1 mL 95% EtOH, vortex, and centrifuge for 10 min, decant supernatant. Dry pellet.