eChapters

Storage

Nucleic acids are very sensitive to contaminations hence care should be taken while processing and storing DNA and RNA. The following properties of reagents and conditions need to be taken care of:

Heavy metals cause breakage of phosphodiester linkage. One can use EDTA which is an excellent heavy metal chelator.

UV rays cause extensive damage to nucleic acids. UV light at 260 nm causes breakage of thymine dimers and cross-link, including loss of biological activities.

Ethidium bromide results in photo oxidation of DNA. Oxidation products can cause phosphodiester breakage.

Nucleases are found on human skin; therefore, it is advisable to avoid direct or indirect contact between nucleic acids and fingers.

Most DNAes are not very stable; however, many RNAses are very stable and can absorb to glass or plastic and remain active.

At -20°C: extensive single and double strand breaks results. Hence for long-term storage it should be stores at -70°C. ? It is best to store DNA in high salt ( >1M) in the presence of high EDTA (>10mM) at pH 8.5, for long-term storage.

Storage of DNA in CsCl with ethidium bromide in the dark is an excellent storage medium. There is about one phosphodiester break per 200 kb of DNA per year.

Storage of ë DNA in the phage is better than storing the pure DNA.

Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids and solids should be autoclaved separately. Ensure that the lids on all bottles are loose. Do not put a large number of items in tray, to ensure that all items reach the appropriate temperature. Also allow sufficient air/steam circulation.

Steps in autoclave

Pressure should read ‘O’ when you open the pressure chamber of the autoclave.

Place items to be autoclaved in the autoclave and close the door. Some autoclaves require that you also lock the door after it’s closed.

Set time - typically 20 minutes.

Temperature should be set at 121°C already, but double-check and change if necessary.

Set cycle: If liquid, set “liquid cycle” or “slow exhaust”. If dry, set “dry cycle” or “fast exhaust” + dry time.

Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to “sterilize”

Several substrates, like proteins (enzymes), are very sensitive to alteration in the concentration of the solution they are placed in. Hence is the importance of a buffer which controls the chemical properties of the medium. Literally a “buffer” has the ability to resist changes in the hydrogen ion concentration. Although, buffers do contain other molecules, that might, in addition to the hydrogen ion concentration, influence the ionic strength, the activity of enzymes, and other parameters of the experiment.

In any biological experiment, appropriate buffer must be selected based on their effect on the experiment. Ideal buffers control pH and ionic strength without interacting adversely with the experiment undertaken. For example, although phosphate is a common buffer, it may not be suited when phosphate is a substrate or product of the reaction being studied. Also, some proteins interact poorly with some buffers. For example, Tris is not ideal as it exhibit high pKa and the large change in pKa when temperature changes, although it is cheap and most proteins are stable in Tris buffers, moreover it seldom reacts with reaction substrates hence, Tris buffer is commonly used in biochemical experiments.

To prepare a series of dilutions of the original more concentrated stock solution, one need to consider both the required final concentration and required volume of the diluted material. The dilution can be readily calculated by the equation:

A spectrophotometer is an analytical instrument that measures the absorbance of a test sample (solution). The term “spectroscopy” comes from the word “spectrum”. “Spectroscopy” therefore implies the use of multiple wavelengths of light. Spectrophotometers have the ability to specifically measure absorbance at specific wavelengths. The most commonly used method to allow this involves a “monochromator”, a device (either a prism, or more commonly, a diffraction grating) that splits the incident light into its component wavelengths, and allows only light of the desired wavelength to reach the sample. The ability to measure absorbance at different wavelengths is very useful, because the extinction coefficient of a compound varies with wavelength. In addition, the absorbance spectrum of a compound can vary depending on the chemical composition of the compound, and depending on the nature of the solvent around the compound.

Absorbance is a useful quantity and follows the Beer-Lambert law. The extinction coefficient of a molecule at a given wavelength can be calculated using the Beer-Lambert equation from absorbance measurements for solutions of known concentration, which states that:

In molecular biology and biochemistry, the ability to accurately and reproducibly measure and transfer small volumes of liquids is critical for obtaining useful results. For volumes less than 1 ml, the most common method for measuring liquid volumes involves the use of a device known as a pipetman or micro-pipette.

The pipetmen can be both fixed and variable volumes. Commonly used pipetmen are: P1000, P200, P20, P10 etc. P1000 are useful for volumes from 200 to 1000 µl. P200 are useful for volumes from 20 to 200 µl. P20 are useful for volumes from 0.5 to 20 µl. For beginners fixed volume pipetmen are more useful. It is important to ascertain whether the correct pipetman is chosen for measuring the volume one needs.

This book is designed to introduce to the students some of the most widely used experimental procedures in biotechnology, including DNA/RNA isolation, manipulation, cloning, protein purification and characterization. The students will also get familiarized with some of the equipments frequently used in biochemistry and molecular biology.

The biotechnology laboratory course is an exploration of procedures. Hence, in order to get full benefit from the course, the students need to read the manual, which is self explanatory. The more effort they put into the course work, the more they will learn. Before the start of actual lab works, one needs to spend some time reading the Laboratory Manual. This reading will provide background information and an outline of the procedures to be performed.

The biotechnology laboratory is conducted as a “directed” research project. This means that although the general procedures are well established, the overall goal of each experiment is the acquisition of new information. Because of the nature of scientific research, predicting the outcome of experiments that have not previously been performed is difficult. It may therefore be necessary to design new experiments based on the results of previous ones, or to repeat experiments that yielded uninterruptable or ambiguous results.

Major Families

This chapeter deals with indigenous knowledge on use of various plant species in traditional medicine, food, dye, wood and in tanning etc.

Malvaceae

The family Malvaceae is also known as Mallow family, characterised by mainly shrubs and herbs. The plants of this family frequent have stellate trichomes, persistant sepals, epicalyx present, stamens numerous and monadelphous, anthers monothecous, carpels 5 or more.

Abroma angusta (Devil’s Cotton)

Paste of the leaf is applied to heal wounds and to relieve joint pain or back pain. Young shoots are used as food. Roots are used to cure itching.

Abutilon indicum (Kanghi)

The whole plant is used to make brooms. Plant also has medicinal value and its sap is used against ulcer, leprosy, piles, dysentery and fever.

India, the land of oldest civilization known through out the world for its vibrant cultures, faiths, lifestyle, traditions, rituals etc. which are interwoven with each other since time immemoral. Here people of different ethnic groups live together who speak different languages and dialects according to their religion and choice. Altogether, 68 million inhabitants belonging to 227 ethnic groups, comprising of 573 tribal communities exists in different part of the country. These people constitute 8.08% of the total population of India (1991 Census of India) and speaks <750 dialects that can be classified into one of the following three language families: Austro-Asiatic (AA), Dravidian (DR), and Tibeto-Burman (TB). When come to think of different tribal community’s inhabiting India, they can be easily divided into six major racial lineages i.e. - Negroid, Mongoloid, Proto-Australoid/ Austrics, Mediterranean or Dravidian, West Brachycephals and Nordic Aryans

Plants have played an important role in many mythologies and religions of the world and have been known for deep and sacred meanings throughout the ages. It is the symbolic representation of the old cultural traditions in the form of a tree across the world. Christmas tree in the Christianity and Bodhi tree in Buddhism are the example of tree featured in mythology. In most of the folklore, folktales and folk religion trees are often said to be homes of tree sprits. If history is traced, we will found that Druidism and Germanic paganism appear to have involved in religious conservational practices of sacred groves. Druidism is a nature based religion that was taught by Druids about 2000 years ago. The term druid itself possibly derives from the religeous word for oak. Therefore, the Druidism reconnects us with nature, our ancesters and ourselves by working with plants or trees.

Trees are honored in Indian culture for their different beneficial characters like medicinal properties, food, fodder, maintaining hydrological cycle, moderate the climate etc. There are some trees that have attained more than a special benevolence in Hindu mythology. In Indian mythology number of plants have been given sacred value and worshipped by different cultures. Some important plants of sacred value have been discussed here.

Before carry out any ethnobotanical work, first define the goal of the assignment or study before selecting the approach, which best suits the interests, budget and schedule. Most of the methods used in ethnobotanical studies are time taking and costly because several field trips are required in several seasons for covering different ritual occasions and seasonal farming activities which are continue for a number of years for completion of an assignment. In this way, an assignment may be very long or short as per selection approach of an ethnobotanist. Long-term assignment or projects may be continue for several years and they require sufficient financial input. When sufficient financial add and time does not available to accomplished long term assignments, in that case a Rapid Ethnobotanical Appraisal (REA) can be done.

Indigenous culture

Indigenous means originating where it is found. If knowledge originates from, and is bound to local experiences, taking its local world not perhaps as the only one, but as the most relevant of all they know may be called indigenous. Indigenous knowledge is human lifeexperience in a distinct natural and cultural amalgamation within a unique local and timely setting. It is an authentic appropriation of being, meaning that this very process happens exclusively in a given locality at a given time. In the field of medicine, Chinese culture (indigenous) developed Acupuncture technique. An acupunturist places needles into the skin to balance out the flow of chi which is believed to be the energy that flows through the human body. In theory, Chi runs along 14 pathways called meridains and when a patient is injured, stressed, or ill, their chi is supposedly being blocked. By placing needles in the Meridians the patients chi is thought to be put back into balance. Like Chinese culture, indigenous culture of different places/countries occurs and have their own belief and methods.

Indigenous cultures speak in various ways, for example we could speak from the perspective of our so-called ‘civilizations’ but how do we know that this perception is not contrary to the truth. We could speak on the basis of our traditional religious concepts but again these concepts may in reality be the opposite of what we believe. If we speak about the indigenous from the point of view of an anthropologist we arrive at a cold and empty language, a study that only pays attention to external and superficial matters, in the same way an anatomist analyses our inner organs or our bones but they are still a long way from knowing the reality. We believe that it is better to speak about indigenous cultures on the basis of their own reality, in relation to how they were as much as how they are now because in the course of time from yesterday to today many things have happened that are worth examining.

In the starting of twentieth century, the ethnobotany progressed from the untested compilation of knowledge to a bigger method and abstract reorientation. Nowadays, the ethnobotany practices need a spread of skills such as:

• Training on the identification and preservation of plant specimens
• Training on linguistic and sociology to understand local language and their family structure
• Training on the psychology and cultural or traditional about plants
• Training on pharmecology and medicobotany

Ethnobotanists interact in a very broad array of analysis queries and practices, that don’t lend themselves to straightforward categorization. However, the subsequent headings arrange to describe a number of the key areas of recent ethnobotanical study.

14.1 Introduction

In this chapter we shall describe various simple and easy to fabricate fiber optic sensors. Most of these sensors are based on intensity modulation. Intensity modulated optical fiber sensors have been extensively studied in comparison to sensors based on other modulation techniques. This is because of their several advantages such as simplicity, reliability, flexibility and relatively low cost. Intensity modulated sensors make use of multimode fibers. Multimode fibers have large core and high numerical aperture that make the coupling of light into the fiber easier. The large core is also better suited to propagate high power laser beams. Further, the plastic-clad fused silica multimode fibers are available. Plastic-clad silica multimode fibers have an additional advantage. Their plastic cladding can be removed easily permitting access to the core. The evanescent field in the unclad part has been used for various kinds of chemical and biochemical sensors (see chapter 5). Above all, these fibers are cheap. This chapter is devoted to simple and easy to make both intrinsic and extrinsic type sensors. Sensors utilizing bare-ended fibers have also been described.

13.1 Introduction

Distributed optical fiber sensors are those sensors which monitor a single measurand at a large number of locations or continuously over the path of the optical fiber. The measurand can be a physical or a chemical quantity. The distributed optical fiber sensor generally consists of a common light source, optical fiber to carry light to the sensing probes and a return fiber to carry modulated light to a photodetector. These sensors can utilize any kind of light modulations described earlier. The advantages associated with distributed sensing are reduced number of components and hence the cost, reduced weight and cabling, and electrical passivity. The applications include the measurements of temperature, pressure, strain, current and chemical concentration. If the measurand is monitored continuously along the fiber length then the sensor is called intrinsic distributed sensor. If it is measured at a finite number of locations then the sensor is called quasi-distributed sensor. The quasi-distributed sensor has a number of advantages over intrinsic distributed sensor. In quasi-distributed sensor, spatial resolution is not as important as in intrinsic distributed sensor provided that individual sensors can be clearly resolved. Further, in quasi-distributed sensing, the sensor elements can be tailored in terms of responsivity and range to meet the specific needs of the application. In this chapter we shall describe various kinds of distributed sensing approaches and few optical fiber distributed sensors.

12.1 Introduction

Among a variety of optical evanescent wave techniques, spectroscopies based on the resonant excitation of surface plasmons have matured into a widely accepted surface analytical method used in different fields. Liedberg et al¹ were the first who demonstrated the exploitation of surface plasmon resonance (SPR) for chemical sensing. Since then the SPR sensing principle has received much attention. In this chapter we shall first describe the principle of this technique and then few sensors that have utilized this phenomenon.

11.1 Introduction

In previous chapters we have described fiber optic sensors based on intensity and phase modulations. These sensors have some problems in practical applications that need to be solved. Some of these are fluctuation of light source power, loss in couplers, scattering and absorption effects that affect measurement performance, temperature drifts and vibrations. In comparison to intensity and phase modulated sensors fiber Bragg grating (FBG) sensors have following advantages:

High resolution

Insensitive to intensity fluctuation because the signal obtained from an FBG sensor is encoded directly in wavelength domain.

Provision of well localized sensing region

Linear output as a function of the measurand

The capability of multiplex many sensors along one fiber

10.1 Introduction

There are very few frequency modulated fiber optic sensors. This is because frequency modulation of light occurs under a limited range of physical conditions. The principal one is based on Doppler effect. There are few other circumstances under which the frequency of light is modulated. These include luminescence and Raman scattering. Luminescence has been included in chapter 7under fluorescence based sensors. In this chapter we shall focus on the sensors based on Doppler effect and Raman scattering. In particular, we shall discuss the measurement of blood flow velocity, colloidal particle motion and Raman spectrum using optical fibers.

9.1 Introduction

Many physical parameters such as pressure, temperature, electric current etc. can be measured using polarization-modulated sensors. In these sensors modulation of state of polarization of light in the sensing region by an external stimuli takes place. A variety of physical effects influence the state of polarization of light. These phenomena are Faraday effect, electro-optic effect and photo-elastic effect. Polarization modulated sensors, in some cases, offer significant advantages not only over conventional sensors but also over widely used fiber optic interferometric sensors. These sensors require control over the state of polarization of light entering the sensing region of the fiber. Thus, for these sensors, highly birefringent fibers or single mode fibers are used. In this chapter we shall first discuss magneto/electro-optic effects and then shall describe sensors for the measurement of electric current/ magnetic field, voltage, pressure, temperature etc. utilizing polarization modulation.

8.1 Introduction

Interferometric or phase modulated sensors have received special attention because these are much more sensitive than intensity modulated sensors and can be used over a much larger dynamic range. The sensors rely on the interferometric technique. Generally, the sensor employs a coherent light source such as a laser and two single mode optical fibers. Light is split and coupled into both the fibers. If the measurand perturbs the optical properties of one of the fibers relative to the other, a phase shift occurs. The change in phase can be measured very precisely. For the measurement a sophisticated electronic processing equipment is needed. Thus the interferometric sensors are expensive compare to intensity modulated sensors. There are four kinds of interferometer configurations: Mach-Zehnder, Michelson, Fabry-Perot and Sagnac interferometers. The Mach-Zehnder, Michelson and Sagnac interferometers are the two beam interferometers while in the case of Fabry-Perot interferometer multiple beam interference occurs. In this chapter we shall first describe interference phenomenon and then different kinds of fiber optic interferometers. In the end we shall describe a number of sensors based on interference phenomenon or phase modulation.