eChapters

The motive behind conducting the workshop on Capacity Need Assessment and Development is that the existing Extension Advisory Services (EAS) needs new capacities to respond effectively to the emerging challenges in agricultural development.

Participatory curriculum reorientation awareness-raising workshop for key stakeholders: In the present study focus group discussion and workshops were conducted to know the effectiveness of present curriculum of extension education with different stakeholders (Students UG, PG and Ph.D, Extension teachers, Field extension workers and farmers). Data were collected by using schedules regarding the important contents of present curriculum and most important for extension workers in the present situation to be included in curriculum

Reagents

Nuclease-Free Water: 14µl

10X Restriction Buffer: 2µl

Acetylated BSA (1mg/ml): 2µl

DNA (~1µg): 1µl

Restriction Enzyme (10u): 1µl

Final Volume: 20µl

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)

The concept of capacity development is defined in various ways. Broadly, it is defined as a process by which skills, and knowledge are built, utilized, retained and nurtured with a view to provide an entity responding to a development challenge. Two components that are vital in any plausible definition of the concept are human skills (the ability to perform a task) and institutions (effectiveness and efficiency of organizations, systems, processes and procedures).

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

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

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.

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:

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

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

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:

In the realm of cell culture, the quality and composition of the growth media and reagents play a pivotal role in growth and sustaining cells in vitro. The preparation of these essential components requires precision, attention to detail, and adherence to the strict protocols to ensure optimal cellular growth, viability, and experimental reproducibility. This introduction serves as a foundational overview of the critical aspects involved in the preparation of media and reagents for cell culture experiments. Furthermore, the preparation of media and reagents for cell culture necessitates adherence to stringent quality control measures to ensure consistency and reproducibility across experiments. Validation of media batches, sterility testing, and periodic quality assessments are essential practic

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.

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.

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.

Introduction

Structural changes in host cells by the viral infection is referred as cytopathic effect (CPE) and the responsible virus is called cytopathogenic virus. As a virus hijacks a cell to make copies of itself, the cell goes through distinct changes in its chemistry and structure. These effects are easiest to see in lab grown cell cultures. The severity of the damage caused by the virus depends on the specific virus itself, the type of cell it infects, how many viruses infect each cell (MOI), and other factors.

The early stages of this damage, called cytopathic effects (CPE), can be spotted directly under a microscope without any dyes or special treatments. While various types of CPE exist in living cells, some signs of viral infection, like inclusion bodies, require staining the cells to be visible. We can classify viruses based on how quickly they cause visible damage (cytopathic effect) in cell cultures. Viruses are typically considered slow if it takes 4-5 days to see this effect in cultures with a low starting dose of virus (MOI). On the other hand, viruses are considered fast if the damage shows up in just 1-2 days under the same conditions. It is important to note that at a high MOI all CPE can occur rapidly. So all decisions about rate of CPE appearance should be based on the lowest MOI that produces CPE

5.1 Purpose

To employ a standard protocol to perform agarose gel electrophoresis.

5.2 Principle

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Migration rate of DNA molecule depends on various factors like 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under UV light.

Genotoxicity potential of any environmental pollutants can be investigated using the micronucleus (MN) test. It is among the most sensitive DNA damage indicators and it has been applied to several organisms and tissues for evaluation of environmental contaminants (Lemos et al., 2011). Micronuclei (MN) assay is more rapid and simpler than chromosomal analysis (Gandhi et al., 2003). Compared to the chromosome aberrations, the final result is relatively easily scored and thus requires less time to make an assessment of the clastogenicity of a chemical (Garriott et al. 2002). An in vitro MN assay can detect the clastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction (Corvi et al., 2008). Although, micronuclei constitute well-characterized biomarkers of chromosomal damage (Salazar et al. 2009) but in vitro MN test does not provide information on the origin of micronuclei (Corvi et al., 2008).

Experimental Procedure

Micronuclei assay is carried out in PMNC and splenocytes as per method of Hayashi et al. (1983).