eChapters

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:

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.

Bacteria are morphologically different. This characteristic is helpful in the identification of bacteria. Routine study of bacterial morphology should include examination of stained preparations. Motility should be examined by hanging drop preparation. According to the requirement, certain cell structures may be stained. Stained preparations should be made preferably from fresh agar slant cultures. Smears of Streptococcus should be made from a liquid culture. Morphology of bacteria may be described under the following terms:

1.Shape: Spherical, coccobacillary, short or long rods, filamentous, comma- shaped, curved or spiral forms.

2.Size: Spherical are measured in diameter and rod-shaped in length and breadth. Measurement is expressed in microns (µ).

3.Sides: Straight, concave, bulging, parallel or irregular.

4.Ends: Round, pointed, truncate or concave.

5.Axis: Straight or curved.

6.Arrangement: Singly, in twos, in groups of four or eight, in grape-like bunches, in long chains, or scattered irregularly, may be in bundles, angular arrangements or in Chinese letters.

7.Pleomorphism: Bacteria may differ from their typical shape. Some of the older cultures may depart greatly from their typical forms, and may show spindles, clubs, and filaments, swollen and branching forms. Variation may be both in shape and size.

8.Capsule: May or may not be present. When present, size should be noted. This requires special staining.

Joint supply : Some products or production processes have more than one use. For instance, cows can both provide milk and be eaten.

Keynesian : A branch of economics, based, often loosely, on the ideas of keynes, characterized by a belief in active government and suspicion of market outcomes. 

Quality Adjusted Life Years (QALYs) : Calculated by adjusting the estimated number of life-years an individual is expected to gain from an intervention for the expected quality of life in those years. The quality of life score will range between 0 for death, to 1 for perfect health, with negative scores being allowed for states considered worse than death

X-efficiency : Producing output at the minimum possible cost. This is not enough to ensure the best sort of economic efficiency, which maximises society’s total consumer plus producer surplus, because the quantity of output produced may not be ideal. For instance, a monopoly can be an X-efficient producer, but in order to maximise its profit it may produce a different quantity of output than there would be in a surplus-maximising market with perfect competition.

Years of potential life lost : A measure of the impact of premature mortality on a population, calculated as the sum of the differences between some predetermined minimum or desired life span and the age of death for individuals who died earlier than that predetermined age.

Zero-sum game : When the gains made by winners in an economic transaction equal the losses suffered by the losers.

Proper records can be maintained and individual performance can be noted.

Disadvantages: A number of males should be maintained which is not economical.

Advantages of polygamous mating: Economical because 1 male can be used for 4-5 females. We can use postpartum estrus effectively.

Immediately after parturition after 7 days they come to heat. Breeding can be done 7 days after parturition after separating the young ones. [Weaning age 21 days]

But this is usually not followed because the number of young ones born reduces subsequently.

Disadvantages: Proper records cannot be maintained.

NATIONAL LEVEL FACILITIES OF LABORATORY ANIMALS

• National Laboratory Animal Centre, Central Drug Research Institute, Lucknow.
• National Centre for Laboratory Animal Sciences (NCLAS) National Institute of Nutrition, Hyderabad

INSTITUTIONAL LABORATORY ANIMAL FACILITIES AT

I. FILL UPS

1. Somatic embryo encased with artificial endosperm and protective cover is called _____.
2. The term artificial seed was coined by_____.
3. The artificial seed technology was patented by_____.
4. The most common gelling agent used for artificial seeds is_____.
5. In desiccated system, the artificial seeds are coated with _____.

I. FILL UPS

1. The initial handful of seeds obtained from selected individual plants of a particular variety is called as _____.
2. The panicles collected should be stored in a airtight drum under _____.
3. _____ is the original purified seed stock of a new variety in the hands of a plant breeder.
4. The seeds of a notified variety collected from the evolving institute with all the passport information is considered as _____.
5. Nucleus seed is produced by _____.

I. FILL UPS

1. Seed village is also known as _____.
2. ITC with Zeneca stands for _____.
3. ITC Zeneca has recently changed name to _____.
4. CENSAR stands for _____.
5. _____ is a compact area approach in which the whole village involved in seed production.
6. The concept of seed village is a _____.
7. Seed village concept was started in_____.
8. _____ has guided the experiment in wheat.
9. _____ is a milestone in green revolution.
10. CAPART stands for _____.

I. FILL UPS

1. Green revolution in wheat was started by _____ in Mexico.
2. Wheat revolution in India was started by_____.
3. Isolation distance for wheat seed production is _____.
4. Seed rate for wheat seed production is _____.
5. Wheat is a _____ pollinated crop
6. Seed multiplication ratio for wheat is ranged from _____.
7. Spacing adopted in wheat seed production plot is _____.
8. Average seed yield in wheat is _____.
9. _____ is the critical moisture content for wheat seed storage
10. The minimum pure seed percentage for wheat foundation seed is _____.

I. FILL UPS

1. The inflorescence of sorghum is _____.
2. The pollen viability in sorghum is_____.
3. Spacing adopted in sorghum seed production is _____.
4. Seed rate recommended for sorghum seed production is_____.
5. Seed yield of sorghum is _____.
6. The moisture content of sorghum seed should be _____ during storage.
7. Isolation distance for hybrid sorghum seed production is_____.
8. Sorghum is a_____ pollinated crop.
9. _____ crop is used as a feedstock for production of ethanol.
10. _____ hybridization technique is followed for sorghum.

I. FILL UPS

1. Pearl millet is a_____ pollinated crop.
2. An example for protogyny is_____.
3. The isolation distance for foundation seed production of pearl millet is _____.
4. The spacing adopted for pearl millet is _____.
5. Seed yield of pearl millet is_____.
6. The moisture content of pearl millet seed during storage should be_____.
7. The planting ratio for pearl millet hybrid seed production is _____.
8. Presence of fertile counter part of female plants in the female rows is called _____.
9. In some female plants, fertile anthers may be hanging out only from a portion of the panicle. Such plants are called _____.
10. _____ technique is used for hybrid seed production of pearl millet.

I. FILL UPS

1. Redgram is commonly is known as _____.
2. _____ is the second most important pulse crop after chick pea.
3. Redgram, being a _____ capable of fixing atmospheric nitrogen.
4. Pigeon pea contains_____% protein.
5. Isolation distance of redgram must be_____.
6. The spacing adopted for short duration red gram is _____.

I. FILL UPS

1. Blackgram is commonly known as _____.
2. Adopted spacing for blackgram is _____.
3. Seed rate for blackgram is _____.
4. Moisture content must be reduced to _____ for short term storage of blackgram seeds
5. Isolation distance for blackgram seed crop is _____.

I. FILL UPS

1. The isolation distance for seed crop of greengram must be _____.
2. Spacing adopted for greengram seed production is _____.
3. Seed rate of greengram is_____.

I. FILL UPS

1. Cowpea is a _____ crop.
2. Isolation distance for cowpea foundation seed production is _____.
3. Spacing adopted for cowpea is _____.
4. Seed rate for cowpea seed production is _____.
5. The maximum permitted limit of offtypes for CS is _____.

I. FILL UPS

1. Lab lab is a _____ crop.
2. Spacing adopted for lablab seed production is _____.
3. The isolation distance for lab lab crop is _____.
4. The offtypes permitted for CS in lab lab crop is _____.
5. Seed rate for field lab lab is _____.

I. FILL UPS

1. Soybean is also known as_____.
2. _____ is the largest oil seed crop in the world.
3. Soybean is a_____ crop.
4. The isolation distance for soybean is _____.
5. Spacing adopted for soybean is _____.
6. Seed rate for soybean is _____.
7. _____ extracted from soybean oil used as emulsifier and lubricant.

I. FILL UPS

1. The isolation distance is _____ for foundation seed production of jute.
2. The maximum permitted limit of off types for foundation seed is _____.
3. The water requirement of jute crop is _____.
4. Seed rate recommended for olitorius type of jute is _____.
5. Seed rate recommended for capsularis type of jute is _____.

I. FILL UPS

1. Groundnut contains _____ oil.
2. Groundnut contains _____ protein.
3. Groundnut is a _____crop.
4. Stigma receptivity is for _____ after anthesis.
5. Fertilization occurs about_____ after pollination.

I. FILL UPS

1. _____ is the fastest growing oilseed crop in India.
2. The inflorescence of sunflower is _____.
3. The tiny flowers (or) florets are attached on a platform called _____.
4. Sunflowers in bud stage exhibit _____.
5. _____ is a tuft of hairs on the fruit.

I. FILL UPS

1. _____ seed is one of the oldest condiments the human race has ever known.
2. Sesame seed contains _____ oil and _____ protein.
3. Sesame oil contains about _____ % oleic acid.
4. Isolation distance for gingelly certified seed production is _____.

I. FILL UPS

1. Castor seeds contain _____ % oil.
2. The castor is rich in which triglycerides _____.
3. _____is a toxin present in castor seed.
4. Castor is a _____ crop.

I. FILL UPS

1. _____ is a multipurpose crop that has been grown for orange red dye.
2. Safflower oil contains _____ % linoleic acid.
3. Orange red dye extracted from safflower flowers is called _____.

I. FILL UPS

1. Tomato is a _____ pollinated crop.
2. _____ pigment is responsible for red colour of tomato.
3. Seed rate recommended for tomato is _____.
4. Isolation distance recommended for foundation seeds of tomato is _____.
5. Spacing adopted for tomato is _____.

I. FILL UPS

1. Brinjal is rich in _____ and _____.
2. In brinjal, _____ is the common feature in case of style.
3. Stigma receptivity of brinjal is _____.
4. Pollen viability in brinjal is _____.
5. The isolation distance recommended for brinjal certified seed production is _____.

I. FILL UPS

1. Dehydrated green chilli is a good source of _____.
2. Isolation distance adopted for chilli for foundation seed production is _____.
3. Seed rate of chilli is _____.
4. Spacing generally adopted for chillies are _____.

I. FILL UPS

1. Capsicum belongs to _____ family.
2. _____ active ingredient is present in capsicum.
3. Ideal day temperature for flowering is _____

I. FILL UPS

1. _____ crop stabilizes the blood sugar by curbing the rate at which sugar is absorbed from the intestinal tract.
2. Okra is _____ crop.
3. The isolation distance adopted for okra for foundation seeds is _____.
4. Seed rate of okra is _____.

I. FILL UPS

1. Onion is a bulbous _____ herb.
2. Onion is a _____ pollinated crop.
3. Thin white mass within the seed coat is called _____.
4. _____ method is commonly followed for onion seed production.
5. The minimum isolation distance for foundation seed is _____.

I. FILL UPS

1. _____ vegetable is botanically called as a fruit.
2. Fruit of pumpkin is called _____.
3. The isolation distance for pumpkin foundation seed production is _____.
4. The isolation distance for pumpkin certified seed production is _____.

I. FILL UPS

1. Ashgourd is a _____ crop.
2. Stigma receptivity is for _____.
3. Ashgourd is a _____ pollinated crop.
4. Ashgourd fruit is a _____.
5. The isolation distance for ashgourd certified seed production is_____.

I. FILL UPS

1. _____ vegetable contain anti-diabetic properties.
2. Bittergourd has _____ similar to bovine insulin.
3. Bittergourd is popularly seen as a _____.
4. Bittergourd is a _____ plant.

I. FILL UPS

1. The stigma receptivity of ribbedgourd is_____.
2. Ribbedgourd is a _____ pollinated crop.
   1. Isolation distance for foundation seed production of ribbedgourd is _____.

I. FILL UPS

1. The viability of pollen grains in snakegourd is for _____.
2. Stigma receptivity is for _____hours after opening.
3. The isolation distance for snakegourd foundation seed production is _____.
4. The wild species of snakegourd is _____.

I. FILL UPS

1. The potato is made up of _____ and _____.
2. Potato is a _____ pollinated crop
3. Potato fruit is called as_____.
4. An example of stem tuber is a _____.

I. FILL UPS

1. _____ number of beds measuring 8 m, 0.6 m are sufficient for raising sufficient seedling for one hectare.
2. Seeds are sown singly at _____distance apart in the furrow.

I. FILL UPS

1. _____ and _____ seeds give highest grain yield.
2. Assessment of _____ is one of the basic requirement for quality control of seed under the seed act.
3. The minimum number of seeds for germination test of _____.
4. The purity analysis in groundnut is done with working sample size is _____.

I. FILL UPS

1. _____ is the official measure for controlling the genetic purity of the seed lot.
2. _____ serves as ‘pre-control’ as well as ‘post-control’ for avoiding genetic contaminations.
3. _____ can also be used as a measure to judge the efficacy of the certification agency or the inspector.
4. The minimum population requirement for taking the observations shall be _____ plants.

I. FILL UPS

1. ISTA was founded in _____.
2. ISTA secretariat is located at _____.
3. _____ was founded with the aim to develop and publish standard procedures in the field of seed testing.
4. _____ is one of the leading international journals featuring papers and articles on seed quality and physiology published by ISTA.
5. The technical committees are made up of _____ number of members.

1. VARIETAL SEED PRODUCTION

3 m : Rice, soybean

5 m : Blackgram, greengram, cowpea, field lab lab lab lab

5-10 m : Groundnut

25 m : Tomato