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Have You heard of the chimera of Greek mythology; part lion, part goat, part dragon, was a creature that was eventually slain by the heroBellerophon. This had its counterpart in the Makara of Hindu myth, with the body of a crocodile and the tail of a fish, also reflected in the Western astrological sign of Capricorn

Chimeras  today are no longer mythical nor part of science fiction; modern-day biology through genetic engineering has allowed for the insertion or deletion of part of the genetic code of an animal in order to change that animal’s characteristics, or its phenotype. What have not changed are the public concerns, curiosities, and anxieties about these developments.

The first transgenic mouse had the genome sequence of the Maloney leukemia virus inserted into its genome. Transgenic animals are used extensively in the study of in vivo gene function, as models for human diseases and in the production of biopharmaceuticals. 

To read more http://microamaze.blogspot.in/2016/02/bioethics-in-transgenic-animals.html

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Hello everyone , This section give the information about the different types of PUMPS used in HPLC The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range. Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). 1) Isocratic pump - Delivers constant mobile phase composition;solvent must be pre-mixed;lowest cost pump , mobile phase solvent composition remains constant with time .Best for simple separations .Often used in quality control applications that support and are in close proximity to a manufacturing process . 2) Gradient pump - Delivers variable mobile phase  composition;can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase .mobile phase solvent (“B”) composition increases with time . Best for the analysis of complex samples .Often used in method development for unknown mixtures . Linear gradients are most popular (for example, the “gradient” shown at right) Also pumps can be Classified based on Delivery systems .       • Binary gradient pump – delivers two solvent       • Quaternary gradient pump –four solvents Solvent delivery system Solvents or mobile phases must be passed via column at high pressure (1000 – 3000psi) because the particle size of stationary phase is few µ (5-10 µ).

 To continue reading   http://microamaze.blogspot.in/2016/02/different-types-of-pumps-used-in-hplc.html

HPLC is an abbreviation for High Performance Liquid Chromatography (It has also been referred to as High Pressure LC). HPLC is a separation technique that involves the injection of sample into packed tube, where individual components of the sample are moved down the packed tube with a liquid forced through the column by high pressure delivered by a pump. Mobile Phase:  Liquid Stationary Phase                                                  Separation Mechanism  

  - Solid                                                                            Adsorption    

- Liquid Layer                                                                 Partition  

 - Ion exchange  resin                                                     Ion exchange    

 - Micro porous beads                                                     Size Exclusion    

- Chemically modified resin                                               Affinity

SOLVENTS - miscible combinations of water or various organic liquids (methanol and acetonitrile), buffers or salts to assist in the separation of the analyte components, or compounds such as Trifluoroacetic acid which acts as an ion pairing agent

PRINCIPLE:         Analyte is forced through a column of the stationary phase by pumping a liquid  at high pressure through the column. The sample is retarded by specific chemical or physical interactions with the stationary phase as it traverses the length of the column. The time at which a specific analyte elutes is called the retention time. Pressure increases the linear velocity. Separated components are detected at the exit of this tube by a flow-through device) that measures their amount. An output from this detector is called a “liquid chromatogram”.

MAJOR HPLC COMPONENTS AND THEIR FUNCTIONS …

http://microamaze.blogspot.in/2016/02/high-performance-liquid-chromatography.html

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With love -Dixy Related Articles AGAROSE GEL ELECTROPHORESIS

Polyacrylamide Gel Electrophoresis (PAGE)

Sodium Doedecyl Sulfate 

Polyacrylamide Gel Electrophoresis (SDS PAGE)

2 D -Gel Electrophoresis 

For more information 

The study of medicines derived from natural sources.

The word "pharmacognosy " is derived from the Greek words φάρμακον pharmakon (drug), and γνῶσις gnosis (knowledge).

Defination :

It is defined as  pharmacognosy as "

The study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources"

. It is also defined as the study of crude drugs.                                                                                 OR

"The study of natural product molecules (typically secondary metabolites) that are useful for their medicinal, ecological, gustatory, or other functional properties.

History :

During 19th and  the beginning of the 20th century—"pharmacognosy" was used to define the branch of medicine or commodity sciences which deals with drugs in their crude, or unprepared, form. Crude drugs are the dried, unprepared material of plant, animal or mineral origin, used for medicine.  

As late as the beginning of the 20th century, the subject had developed mainly on the botanical side, being particularly concerned with the description and identification of drugs both in their whole state and in powder form. Such branches of pharmacognosy are still of fundamental importance, particularly for pharmacopoeial identification and quality control purposes, but rapid development in other areas has enormously expanded the subject.

Although most pharmacognostic studies focus on plants and medicines derived from plants, other types of organisms are also regarded as pharmacognostically interesting, in particular, various types of microbes (bacteria, fungi, etc.), and, recently, various marine organisms.

Fields under Pharmacognosy:

Medical ethnobotany: the study of the traditional use of plants for medicinal purposes;

Ethnopharmacology: the study of the pharmacological qualities of traditional medicinal substances;

Phytotherapy:  the medicinal use of plant extracts

Phytochemistry: the study of chemicals derived from plants (including the identification of new drug candidates derived from plant sources).

Zoopharmacognosy: the process by which animals self-medicate, by selecting and using plants, soils, and insects to treat and prevent disease.

Marine pharmacognosy: the study of chemicals derived from marine organisms.

To read more 

http://microamaze.blogspot.in/2016/01/pharmacognosy.html

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Hello everyone,      

Today I am discussing a different method for quantification of DNA ie Quantifying DNA in Solution with PicoGreen

  I have covered similar topics earlier....

DNA QUANTIFICATION USING MINIGELS

Estimating the concentration of DNA by Fluorometry using Hoechst 33258

PicoGreen dsDNAQuantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biological procedures such as cDNA synthesis for library production and DNA fragment purification for subcloning, as well as diagnostic applications, such as quantitating DNA amplification products and primer extension assays.

The PicoGreen assay allowed the detection of 25 pg/ml dsDNA , surpassing the sensitivity achieved with Hoechst 33258 by 400-fold. The assay is most quickly and easily performed using a handheld fluorometer.

The standard PicoGreen assay protocol is also simpler than the Hoechst 33258 method, because a single concentration of the PicoGreen Reagent allows detection over the full dynamic range of the assay.Unlike hoechst – based assay, assay using a single concentration of picogreen are linear over four orders of magnitude in DNA concentration from 1ng/ml to 1 µg/mL.

  PRINCIPLE:-

PicoGreen  dsDNA Quantitation Reagent is an ultra-sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) in molecular biology procedures.The brightness of this reagent is due to its high quantum yield and large molar extinction coefficient. These procedures include cDNA synthesis for library production, DNA fragment purification for sub cloning, and diagnostic applications, such as quantitating DNA amplification products,  and primer extension assays.

  REAGENTS

       Picogreen dsDNA quantitation reagent

       Reference DNA (eg calf thymus DNA) at concentration of 2 µg/mL  in TE(pH 7.5)

        TE (pH 7.5)

To read more http://microamaze.blogspot.in/2016/01/quantifying-dna-in-solution-with.html 

Other methods for DNA Quantification DNA QUANTIFICATION USING MINIGELS Estimating the concentration of DNA by Fluorometry using Hoechst 33258 Extraction and Purification of Plasmid DNA by Alkaline Lysis MethodEstimation of DNA by Diphenylamine method

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Hello everyone,Today I am going to write about one of the methods used to determine Concentration of DNA ie estimating the concentration of DNA by Fluorometry using Hoechst 33258. I have already covered some methods to determine concentration of DNA

AGAROSE GEL ELECTROPHORESIS, Estimation of DNA by Diphenylamine method , DNA QUANTIFICATION USING MINIGELS

INTRODUCTION:-

Quantitation of DNA is a prelude to many practices in Molecular Biology. Accurately determining the DNA concentrations of crude chromosomal or purified plasmid DNA samples is not only desirable, but an essential step in quantitative manipulations of DNA. Common techniques that use DNA, such as sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (e.g. Random prime labeling), etc., all benefit from a defined template concentration. Failure to produce results from these techniques can sometimes be attributed to an incorrect estimate of the DNA template used. DNA concentration is measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1cm path length cuvette.

PRINCIPLE:-

Hoechst 33258 (bisbenzimidazole ) is indol derived stain in free solution has maximum at 356 nm and emission maximum at 492nm . Hoechst 33258 only bind efficiently to small fragments of DNA. Concentration of Hoechst 33258 in reaction mixture should be kept low (5X10-7 M to 2.5X10-6). All DNAs and solutions should be free of ethidium bromide. Hoechst 33258 dye is cell permanent.

Fluorometry assay with Hoechst 33258 do not work at extremes of pH and are affected by both detergents and salts. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm.

The use of Hoechst 33258 in conjunction with the fluorescence microplate reader offers high specificity, as well as high sensitivity for ds DNA quantitation. The concentration of DNA in unknown sample is estimated from std curve constructed using set of std dna (0-20  µg/mL   ) whose base composition is Same as unknown.

REAGENT :-

DNA of known concentration required to construct a std curve

Florometry buffer :- 2MNaCl , 50 mM sodium phosphate (pH7.4). Prepare 500mL and srerlize through a 0.45  filter

High molecular weight DNA solution (100 µg/ml, in TE pH 8.0)

Hoechst 33258 dye (1 mg /ml in water)

Test DNA sample.

EQUIPMENT:-

Low fluorescent background

Black , U shaped ,96- Microfluor B plates

Plate reader

 To read more  http://microamaze.blogspot.in/2016/01/estimating-concentration-of-dna-by.html

With love

-Dixy

What Exactly is a chromosome?

Chromosomes are the rod-shaped , filamentous bodies present in the nucleus , which become visible during cell division . They are the carriers of the gene or unit of heredity. Chromosome are not visible in active nucleus due to their high water content , but are clearly seen during cell division.

The eukaryotes  there are 3 the usual type of chromosomes in their body cells,  but may also contain some unusual and special types of chromosomes in some body cells or at some particular stage of their life cycle. The special type of eukaryotic chromosomes are following:

1)Lamp brush chromosomes:

These are the largest known chromosomes found in the yolk rich oocytic nuclei of certain vertebrates such as fishes, amphibians, reptiles and birds.

seen with naked eye and are characterized by fine lateral loops, arising from the chromomeres, during first prophase (diplotene) of meiosis.

Germline chromosome with lat­erally projecting loops is termed as lamp brush chromosomes. This name was given because it is similar in appearance to the brushes used to clean lamp chimneys in centuries past.

These are found in , frog, toads, salamanders

The bristle are loops of unwound DNA with many RNA polymerase and mRNA molecule

In diplo- tene stage of meiosis, the yolk-rich oocytes of vertebrates con­tain the nuclei with many lamp brush-shaped chromosomes of exceptionally large sizes.

2)Giant chromosomes or salivary gland chromosomes or  Polytene chromosomes :

Found in the larvae of certain dipterans. Occur in the larval salivary glands, midgut epithelium, and rectum and Malpighian tubules of various genera (Drosophila, Sciara, Rhynchosciara, and Chironomus)

Balbiani in 1881 was first observed salivary gland chromo­somes in the salivary glands of dipteran hence it is called as salivary gland chromosomes.

Due to presence of many chromonemal fibrils on them these chromosomes are termed as polytene chromosomes by Koller.

During larval development, specific areas on polytene chromosomes become uncoiled, forming localized regions called ‘pufs’

With love

- Dixy

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