Laminar Air-Flow Cabinets

1. It is highly improved sophisticated inoculation chamber, used for aseptic transfer for microbial cultures and reducing the danger of infection.

2. It is designed in such a manner that sterile materials remain protected from contamination.

3. Laminar air flow works on the principle of air filteration through very fine HEPA filters (High Efficiancy Particulate Air) and single direction air flow.

4. On the working bench of laminar air flow, filtered room air is flowed in a single direction.

5. Laminar flow bench are available in two systems. (A) Horizontal (B) Vertical air flow systems.

6. The highly sophisticated instrument of the lab. is equipped with following attachments fiberglass filters, blowers, UV tube light, fluorescent tube for illumination, glass doors.

Autoclave (Moist Heat Sterilizer)

1. It is an instrument designed on the working principle of steam pressure cooker.

2. It is constructed with that of heavy gauge metallic sheet, so that it can sustain internal steam pressure when in use.

3. In an autoclave saturated moist heat is circulated, hence normally heat labile substances (media, cotton, gauges, liquids and clothing material) are sterlized with it.

4. Each autoclave is equipped with separating thick lid (door) bearing rubber gas kit, and having facility for pressure measure and inner pressure release system.

5. In an autoclave normally 15-pounds pressure is maintained for 15-20 minutes to achieve sterlization of kept material.

6. An electric water heating element (200 watt) is fitted at the base of the autoclave body for boiling placed inside water.

7. All the materials for sterlization are kept in separate container on a stand or wire mass supplied with autoclave.

8. All the internal air of the autoclave chamber should be allowed to escape before closing the steam release cock, and to start the sterlization.

9. When sterilization is completed the light current switch should be brought to off position, and allowed to    release pressure by taking its own time. In any condition pressure release valve should not be opened.

Genomics Applications

1. Fighting disease:

Some human diseases and defects are directly or indirectly caused by genetic abnormalities. Sickle cell anemia, for example, is caused by a change in just one nucleotide out of six billion. Specific genes have been associated with breast cancer, deafness, and blindness. Some illnesses are caused by complex, interacting environmental and genetic factors and cannot be explained by classical inheritance patterns. Genome studies help medical researchers understand the molecular details of these diseases so they can pursue innovative drug treatments and more quickly identify high-risk individuals who could benefit from early medical intervention. And the analyses of the genomes of disease-causing microbes, viruses, and insects, such as the human malaria parasite and its carrier, the Anopheles mosquito, are helping in the development of new prevention and treatment strategies.

Example: Most cystic fibrosis is from one DNA mutation — deletion of just 3 nucleotides — causing buildup of large amounts of mucus in the lungs.

2. Protecting plant life:

Fungi and other plant pathogens cause billions of dollars in damage every year to agricultural crops, plants, and trees. Sequencing their genomes is helping botanists and foresters find effective treatments. Better understanding of plant genetics is also improving crop yields and enhancing the nutritional value of food.

Example: An oak tree damaged by the sudden oak death pathogen Phytophthora ramorum.

3. Harnessing nature’s technology:

Microbes — nature’s simplest and most abundant organisms — can thrive under extreme conditions of heat, cold, pressure, and even radiation. By studying their genomes, scientists hope to find ways to use bacteria and other microorganisms to solve a variety of environmental problems, develop new energy sources, and improve industrial processes. Some microbes can help clean up hazardous waste sites by absorbing, transforming, or breaking down contaminants — a technique called bioremediation. Others can help combat global warming by absorbing, or sequestering, carbon from the atmosphere. And microbes can convert a wide range of organic and inorganic materials into renewable energy. 

Example: The bacterium Rhodopseudomonas palustris can degrade complex aromatic hydrocarbons, assimilate carbon, and provide insights into biomass and biofuel production, particularly hydrogen.

4. Human differences and mutations:

The DNA Sequence in every human is 99.9 percent identical to that of every other human. The slight variations in our genomes are called single nucleotide polymorphisms, or SNPs. Scientists estimate that there are about 1.4 million locations on the genome where SNPs occur in humans. It is these small variations that contribute to individual differences. SNPs and other mutations can be caused by copying errors as DNA is reproduced, or triggered by radiation, viruses, or toxic substances in the environment.

5. Understanding DNA:

Comparing the DNA sequence patterns of humans side-by-side with those of wellstudied “model organisms” such as the fruit fly, mouse, pufferfish, and sea squirt is one of the most powerful strategies for identifying human genes and determining how they’re regulated and what they do. Conserved sequences—DNA patterns that we share with other organisms—are likely to have important functions or they would have disappeared as the organisms evolved.

The analysis of similar segment of DNA is in the genomes of the human, gorilla, pig, rabbit, mouse, rat and chicken and visualizations make it easier for scientists to identify conserved regions of DNA that could be important in regulating gene and protein function.

6. Functional Non Coding Sequence finding:

Along with helping identify genes and their functions, comparative genomics is shedding light on the functions of the noncoding sequences of DNA found within and between the genes. These segments can regulate gene expression, the process involved in determining when and where in the organism a given gene is turned on or off. Understanding the complex orchestration of gene and protein networks is a crucial aspect of contemporary biomedical research.

Posted By:

Mitesh Jain.

How To Prepare MURASHIGE-SKOOG Medium

Murashige-Skoog (MS) medium is widely used for plant tissue culture because it has proven effective for growth promotion of both monocotyledons and dicotyledons.This medium is characterized by high  concentration of mineral salts, nitrate and ammonium which appears to be preffered by cells of some species.

Ingredients:

Macronutrients:

Ammonium nitrate (NH4NO3) 1,650 mg/l

Boric acid (H3BO3) 6.2 mg/l

Calcium chloride (CaCl2 · 2H2O) 440 mg/l

Cobalt chloride (CoCl2 · 6H2O) 0.025 mg/l

Magnesium sulfate (MgSO4 · 7H2O) 370 mg/l

Cupric sulfate (CuSO4 · 5H2O) 0.025 mg/l

Potassium phosphate (KH2PO4) 170 mg/l

Ferrous sulfate (FeSO4 · 7H2O) 27.8 mg/l

Potassium nitrate (KNO3) 1,900 mg/l

Manganese sulfate (MnSO4 · 4H2O) 22.3 mg/l

Potassium iodide (KI) 0.83 mg/l

Sodium molybdate (Na2MoO4 · 2H2O) 0.25 mg/l

Zinc sulfate (ZnSO4·7H2O) 8.6 mg/l

Na2EDTA · 2H2O 37.2 mg/l


Common organic additives:

i-Inositol 100 mg/l

Niacin 0.5 mg/l

Pyridoxine · HCl 0.5 mg/l

Thiamine · HCl 0.1 mg/l

IAA 1–30 mg/l

Kinetin 0.04–10 mg/l

Glycine (recrystallized) 2.0 g/l

Edamine S 1.0 g/l

Sucrose 20 g/l

Agar 10 g/l

REQUIREMNETS: Constituents of the MS medium, Erlenmeyer flasks (100, 250, 500 ml, 1 litre capacity), measuring cylinders (100, 1000 ml capacity), pipettes (1, 5, 10 ml), distilled or demineralized water, PH meter, 1.0 N NaOH, HCl or KOH and autoclave.


PROCEDURE:

1. Prepare macronutrients solution in 100 ml distilled water.

2. Prepare stock solution dilution chard.

3. Add macronutrients in 1-litre Erlenmeyer flask.

4. Add the other heat stable constituents (ex. sucrose, vitamins and harmones) and agar powder(if desired at a concentration of 0.8-1.0%).

5. Make the final volume of the medium by the addition of more distilled water.

6. Adjust pH of the medium to 5.7, using 0.1 N NaOH or 0.1 N HCl.

7. If solid medium is desired, agar is used.

8. Pour the medium into the desired culture vessels (15 ml in 125 * 150 mm culture tube and 50 ml in a 250 ml flask).

9. Plug the culture vessels with non-absorbent cotton wool wrapped in cheese-cloth or with any other suitable closure.

10.Transfer the culture vessels to appropriate baskets covered with aluminium foil to check wetting of plugs during autoclaving.

11.Transfer the baskets to autoclave.

12.Sterlize the medium by autoclaving at 121`c for the time period depending upon the volume of the medium in the vessel.

13.The medium is allowed to cool at room temperature and should be stored at 4 `c for future use.

Posted by:
Mitesh Jain.

Measurement Of Plant Cell Growth

OBJECT: To learn the basic techniques for calculating and plotting plant cell growth.

MATERIALS: MEDIA: BM medium

                      REAGENTS: Distilled water, Ethanol 95% solution, Lugol's solution

                       

                      EQUIPMENTS: Sterile 1 ml pipettes, Sterile 10 ml pipettes, Fuchs-Rosenthal or  eubauer hemocytometers, handheld or mechanical counter,Pandorina morum algae cell culture, Light microscope, Lintless tissues, Graph paper.

PROCEDURE:

(1). Using a sterile 1 ml pipette, inoculate a BF culture vessel with 1 ml of pandorina morum culture.

(2). Shake the culture vessel for 15 sec to evenly suspend the cells.

(3). Incubate the culture at room temperature on an orbital or reciprocal shaker under cool-white fluorescent lights.

(4). Measure the cell density of the culture every day for 2 weeks at approximately the same time each day.

(5). Practice good technique, and avoid contaminating the algae cultures.


Posted By:
Mitesh Jain.

                      

                      

Comparative Genomics

Comparative genomics is an exciting new field of biological research in which the genome sequences of different species - human, mouse and a wide variety of other organisms from yeast to chimpanzees - are compared.

Comparative genomics is the analysis and comparison of genomes from different species. The purpose is to gain a better understanding of how species have evolved and to determine the function of genes and noncoding regions of the genome. Researchers have learned a great deal about the function of human genes by examining their counterparts in simpler model organisms such as the mouse. Genome researchers look at many different features when comparing genomes: sequence similarity, gene location, the length and number of coding regions (called exons) within genes, the amount of non-coding DNA in each genome and highly conserved regions maintained in organisms as simple as bacteria and as complex as humans.

Comparative genomics involves the use of computer programs that can line up multiple genomes and look for regions of similarity among them. Some of these sequence-similarity tools are accessible to the public over the Internet. One of the most widely used is BLAST, which is available from the National Center for Biotechnology Information (NCBI). BLAST is a set of programs designed to perform similarity searches on all available sequence data.

Comparative Genomics of Human and Mouse:

Mice and humans (indeed, most or all mammals including dogs, cats, rabbits, monkeys, and apes) have roughly the same number of nucleotides in their genomes - about 3 billion base pairs. This comparable DNA content implies that all mammals contain more or less the same number of genes.

The most significant differences between mice and humans are not in the number of genes each carries but in the structure of genes and the activities of their protein products. Gene for gene, we (Human being) are very similar to mice. What really matters is that subtle changes accumulated in each of the approximately 30,000 genes add together to make quite different organisms. The following phenomena make different two organisms different.

(1)  Genes and proteins interact in complex ways that multiply the functions of each.

(2)  A gene can produce more than one protein product through alternative splicing.

(3) Post-translational modifications events in each organism occur at different circumstances and these events

     do not always occur in an identical way in the two species

(4) A gene can produce more or less protein in different cells at various times in response to developmental or   

     environmental cues.

(5) Many proteins can express disparate functions in various biological contexts.

Thus, subtle distinctions are multiplied by the more than 30,000 estimated genes.

The often-quoted statement that we share over 98% of our genes with apes (chimpanzees, gorillas, and orangutans) actually should be put another way. That is, there is more than 95% to 98% similarity between related genes in humans and apes in general. (Just as in the mouse, quite a few genes probably are not common to humans and apes, and these may influence uniquely human or ape traits.) Similarities between mouse and human genes range from about 70% to 90%, with an average of 85% similarity but a lot of variation from gene to gene (e.g., some mouse and human gene products are almost identical, while others are nearly unrecognizable as close relatives). Some nucleotide changes are “neutral” and do not yield a significantly altered protein. Others, but probably only a relatively small percentage, would introduce changes that could substantially alter what the protein does.

Put these alterations in the context of known inherited human diseases: a single nucleotide change can lead to inheritance of sickle cell disease, cystic fibrosis, or breast cancer. A single nucleotide difference can alter protein function in such a way that it causes a terrible tissue malfunction. Single nucleotide changes have been linked to hereditary differences in height, brain development, facial structure, pigmentation, and many other striking morphological differences; due to single nucleotide changes, hands can develop structures that look like toes instead of fingers, and a mouse's tail can disappear completely. Single-nucleotide changes in the same genes but in different positions in the coding sequence might do nothing harmful at all. Evolutionary changes are the same as these sequence differences that are linked to person-to-person variation: many of the average 15% nucleotide changes that distinguish humans and mouse genes are neutral; some lead to subtle changes, whereas others are associated with dramatic differences. Add them all together, and they can make quite an impact, as evidenced by the huge range of metabolic, morphological, and behavioral differences we see among organisms.


Posted By:
Mitesh Jain.

Genome Sizes Of Humans And Other Organsims

Comparative genome sizes of humans and other organisms

Organism	estimated size	estimated gene number	average gene density	chromosome number
Homo sapiens (human)	2900 million bases	~30,000	1 gene per 100,000 bases	46
Rattus norvegicus (rat)	2,750 million bases	~30,000	1 gene per 100,000 bases	42
Mus musculus (mouse)	2500 million bases	~30,000	1 gene per 100,000 bases	40
Drosophila melanogaster (fruit fly)	180 million bases	13,600	1 gene per 9,000 bases	8
Arabidopsis thaliana (plant)	125 million bases	25,500	1 gene per 4000 bases	10
Caenorhabditis elegans (roundworm)	97 million bases	19,100	1 gene per 5000 bases	12
Saccharomyces cerevisiae (yeast)	12 million bases	6300	1 gene per 2000 bases	32
Escherichia coli (bacteria)	4.7 million bases	3200	1 gene per 1400 bases	1
H. influenzae (bacteria)	1.8 million bases	1700	1 gene per 1000 bases	1

Genome size does not correlate with evolutionary status, nor is the number of genes proportionate with genome size.


Posted By:
Mitesh Jain.