How to isolate a plasmid from a bacteria ?

How to isolate a plasmid from a bacteria ?

Plasmids are easily isolated from bacterial cells Plasmid isolation takes advantage of the unique structural properties of plasmids. Plasmids are small,super coiled circular pieces of Dana.Unlike the much larger bacterial chromosomes(which is also circular),plasmids are quite resistant to permanent den. Today,most laboratories use commercial kits for plea mid isolations,because the kits are convenient and relatively inexpensive.the kits give good yields of highway a little,while avoiding the need for organic denaturant ants.A variety of less expensive,but somewhat more time-consuming,procedure have been described for investigators who want to make their own reagents.These procedures generally give good yields of death at is slightly less pure in a purified with the kits.Whatever the isolation procedure,the general principles of plasmid isolation are the same.The figure and paragraphs below summarise regs general principles used for plasmid isolation.

1. Lysis and denaturation-

Strong denaturating conditions to weaken the tough bacterial drop wall.The most common procedures use a combination of strong base and a detergent.The detergents help to solubility lipids in the,allowing the denaturants to enter the cell. Proteins,because of their fragile structures,are irreversibly denatured.The treatment also breaks the hydrogen bonds holding together the chromosomal and plasmid DNA.

 2. Neutralization-

Neutralization allows complementary DNA strand store anneal and causes proteins to precipitate.Plasmids renature because they have super coiled structures that have held the two strands of the helix together denaturation.chromosomal DNA a is not able to renature,however,because it's longer strands have become mixed with denatured proteins. Samples must be mixed gently at this step to prevent fragmentation of the long, chromosomal DNA into pieces that might be able to reanneal and co-purify with the plasmids.  

3. centrifugal- 

on plasmid Dana is separated from large aggregates of precipitated protea chromosomal in a by centrifugal ion. 

4. Additional purification -

Plasmids are further purified by organic extraction or adsorption in resin.

What is plasmid DNA?

What is plasmid DNA?

=>

Plasmid is a double standed ,self replicating,extra chromosomal DNA Plasmids are cloning vectors that are widely used in molecularbiology andthey play an important roles in the laboratory.Plasmids are small, circular pieces of DNA  that replicate independently of the host chromosome.The first plasmids used in the lab were derivatives of naturally I occurring plasmids found in vacate.Since their discovery,molecularbiologists have added many features to plasmids to suit a variety of applications.In this lab,each team will I so late three plasmids from bacterial strains.

Plasmid DNA are red circular one

a short notes on Insulin of human body

                                       Human Insulin 

Human insulin is a globular protein with a molecular weight of about 5,800 kd, consisting of 51 aminoacid residues organised in two polypeptide chains (A and B), linked by two disulphide bonds. Chain A consists of 21 residues with an extra disulphide bond between A6 and A11; chain B consists of 30 aminoacids. Complete synthesis of the human insulin molecule was achieved in 196610. Insulin exists as a monomer only at low concentrations while it shows propensity to aggregate into stable dimers at higher concentrations, in aqueous solution at pH 2-8 and into hexamers in the presence of zinc ions. The hexamer, in which chain A constitutes much of the polar surface, is almost spherical in structure, with a diameter of 5 nm and a height of 3.5 nm. Polymerisation of the hormone has major pharmacological implications.

What Is Cancer?

 What Is Cancer?

=> Cancer results from a series of molecular events that fundamentally alter the normal properties of cells. In cancer cells the normal control systems that prevent cell overgrowth and the invasion of other tissues are disabled. These altered cells divide and grow in the presence of signals that normally inhibit cell growth; therefore, they no longer require special signals to induce cell growth and division. As these cells grow they develop new characteristics, including changes in cell structure, decreased cell adhesion, and production of new enzymes. These heritable changes allow the cell and its progeny to divide and grow, even in the presence of normal cells that typically inhibit the growth of nearby cells. Such changes allow the cancer cells to spread and invade other tissues. The abnormalities in cancer cells usually result from mutations in protein-encoding genes that regulate cell division. Over time more genes become mutated. This is often because the genes that make the proteins that normally repair DNA damage are themselves not functioning normally because they are also mutated. Consequently, mutations begin to increase in the cell, causing further abnormalities in that cell and the daughter cells. Some of these mutated cells die, but other alterations may give the abnormal cell a selective advantage that allows it to multiply much more rapidly than the normal cells. This enhanced growth describes most cancer cells, which have gained functions repressed in the normal, healthy cells. As long as these cells remain in their original location, they are considered benign; if they become invasive, they are considered malignant. Cancer cells in malignant tumors can often metastasize, sending cancer cells to distant sites in the body where new tumors may form.

What is a species?

What is a species?

=>

 The most common definition is based on the biological species concept. By this definition, individuals belong to the same species if they reproduce by mating with each other and are reproductively isolated from other such groups.

How to prepare ACETIC ACID- SODIUM ACETATE BUFFER?

(This is practical experiment)

How to prepare ACETIC ACID- SODIUM ACETATE BUFFER?

=>

 REAGENTS REQUIRED:  

Acetic Acid 0.2M: 1.5 ml of glacial acetic acid is made upto 100ml with     distilled water. 

Sodium Acetate Solution: 0.64 gm of sodium acetate or 2.72gm of sodium acetate trihydrate is dissolved in 100ml Distilled water. 

PROCEDURE: 

Pipette out exactly 36.2ml of sodium acetate solution into 100ml of standard flask and add 14.8ml of glacial acetic acid, make the volume 100ml using distilled water using distilled water. This gives 0.2 M of acetic acid and sodium acetate buffer. The pH is measured with pH meter.  

 pH meter is first standararised with pH buffer. Wash electrode with distilled water and introduced into 0.2M acetic acid-sodium acetate buffer prepared, the pH of solution is 4.6. 

 RESULT:  

36.2ml Sodium acetate and 14.8 ml glacial acetic acid were mixed and buffer was prepared. pH was measured initial reading observed was 4 which made upto 4.6 with 5N NaOH.   

How to prepare BARBITONE BUFFER?

(This is practical experiment)

How to prepare BARBITONE BUFFER?

  REAGENTS REQUIRED:

• Diethyl barbituric acid. 

• Sodium diethyl barbititrate 

PROCEDURE:   

Dissolve 2.85gm of diethyl barbituric acid and 14.2gm of sodium diethyl barbititrate in distilled water and upto 1 liter. This gives the barbitone buffer.  The pH meter is first standararised with pH buffer. Wash electrode with distilled water and introduced into barbitone buffer prepared, the pH of solution is 6.8. 

What is CITRATE BUFFER? How it to be made?

    (This is practical experiment)

What is CITRATE BUFFER? How it to be made?

  => 

REAGENT S REQUIRED: 

• Citric acid: Dissolve 2.101 gm of citric acid in 100ml distilled water.

 • Sodium citrate solution 0.1 M: Dissolved 2.941gm of sodium citrate in 100ml distilled water. 

PROCEDURE:  

46.5ml of citric acid with 3.5ml of sodium citrate solution  and upto 100ml with distilled water. It corresponds to 0.1 M citrate buffer and standardised with pH meter and measures the pH of the prepared solution. This gives citrate buffer at pH 2.5. 

RESULT: 

Citrate buffer was prepared and the pH observed was 4.8 which was adjusted to 2.5 using 1N Hcl and 5N NaoH.  

How to make CARBONATE- BICARBONATE BUFFER?

        ( This is a practical experiment)

How to make CARBONATE- BICARBONATE BUFFER?

=>

 REAGENTS REQUIRED:

 • Sodium carbonate solution 0.2M: Dissolve 2.12gm of anhydrous sodium carbonate in 100ml Distilled water.

 • Sodium bicarbonate solution: Dissolve 1.68gm of sodium bicarbonate in 100ml of distilled water.

PROCEDURE: 

Pipette out exactly 27.5ml of sodium carbonate (Na2Co3) solution. To this add 22.5ml of sodium bicarbonate solution and made upto 100ml with distilled water which corresponds to 0.2 M sodium carbonate and bicarbonate buffer. Standardise pH meter and measure the pH of required buffer. This gives the Carbonate- bicarbonate buffer pH 10.2. 

RESULT: 

Carbonate bicarbonate buffer was prepared and pH observed was 7.5 which was adjusted to 10.2 using 1N Hcl and 5N NaoH. 

How to make PHOSPHATE BUFFER?

How to make PHOSPHATE BUFFER?

  =>
REAGENTS REQUIRED:

• Monobasic: Dissolve 2.78gm of sodium dihydrogen phosphate in 100ml of distilled water.

• Dibasic sodium phosphate (0.2M): Dissolve 5.3gm of disodium hydrogen phosphate or 7.17 gm sodium hydrogen phosphate in 100ml distilled water.

PROCEDURE:
39 ml of dihydrogen sodium phosphate is mixed with 61 ml of disodium hydrogen phosphate This made up to 200ml with distilled water .This gives phosphate (Po4)2 buffer of 0.2M. Standardized pH meter with standard buffer. Washed electrode with distilled water and introduced it into phosphate buffer prepared. The pH of the solution is 6.8.

RESULT:
Phosphate buffer was prepared and pH was observed 8.5 which was made upto 6.8 using 1N Hcl and 5N NaoH. 

what is phosphatase?

What is phosphatase?

=>Phosphatases are enzymes that hydrolyze phosphate groups from a wide variety of organic substrates (phosphate esters), producing an alcohol and phosphoric acid. They are found in all cells and usually are classified as either acid phosphatases or alkaline phosphatases.  Phosphatases can be studied in crude cellular extracts or in pure form.

Substrate+Water+Enzyme=Product+Phosphoric acid+Enzyme 

what is enzyme?

• What is ENZYMES?
• How it work on body?   
• => A complex protein produced by living cells that promotes a specific biochemical reaction by acting as a catalyst. Enzymes are catalysts, speeding up chemical reactions in cells, making reactions run to completion thousands if not millions of times more quickly than if the enzyme was absent. 
•  For example, carbonic anhydrase, which functions in red blood cells, is an enzyme that converts 600,000 molecules of carbon dioxide to 600,000 molecules of bicarbonate in ONE second.
•   The rate at which different enzymes convert substrate(s) to product(s) is highly variable, and this rate is also subject to change due to environmental conditions, such as temperature and the pH of the surrounding environment.  For example, rates of decomposition (a complex process whereby myriad chemical reactions occur, with associated enzymes) are much slower in colder climates than in warmer climates decomposition is also slowed if not stopped all together in the highly acidic environment of bogs. 
• An enzyme works on a substrate, which is then turned into a product (or products).  In one of the examples above, carbonic anhydrase is the enzyme, carbon dioxide is the substrate it works on, and bicarbonate is the resulting product.  In text, enzymes are easily recognizable, as they usually end in the suffix –ase. 

What is the Fundamental Properties of Life?

What is the Fundamental Properties of Life?

=>all known organisms share certain general properties. To a large degree, these properties define what we mean by life. The following fundamental properties are shared by all organisms on earth.

Cellular organization-All organisms consist of one or more cells—complex, organized assemblages of molecules enclosed within membranes  Sensitivity.All organisms respond to stimuli— though not always to the same stimuli in the same ways.

Growth-All living things assimilate energy and use it to grow, a process called metabolism. Plants, algae, and some bacteria use sunlight to create covalent carboncarbon bonds from CO2and H2O through photosynthesis. This transfer of the energy in covalent bonds is essential to all life on earth.

Development-Multicellular organisms undergo systematic gene-directed changes as they grow and mature.

Reproduction-All living things reproduce, passing on traits from one generation to the next. Although some organisms live for a very long time, no organism lives forever, as far as we know. Because all organisms die, ongoing life is impossible without reproduction.

regulations- All organisms have regulatory mechanisms that coordinate internal processes.

Homeostasis- All living things maintain relatively constant internal conditions, different from their environment.

What is Biological diversity ??

=>Evolution results in a vast number of different adaptations for survival and reproduction. Life exists in many different forms in soils and on the surface of the land, in water and in the air. All species on earth have not yet been discovered. Fully 1.8 million species are known today. There may be as many as 100 million! About one million of currently known species are insects. There are at least 360,000 green plants. Another 75,000 are molluscs (octopi and squids, snails, mussels, etc.) Nearly 60,000 fungi have been scientifically described. Among the vertebrates, roughly 28,500ray-finned fishes, 10,000 birds, 8,000 reptiles and 5,000 mammals have been described. There are at least 4,000 species of red algae. Knowledge of species and their relationships to each other is essential to much biological research.

What Is Life?

=>Life is nothing but the compostion of hdrocarbon organic metrial .
All known organisms share certain general properties, and to a large degree these properties define what we mean by life.