BIO TECH KITS

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Most of the protocol consists of 2 steps, first lysing the cell and solubilizing the DNA, which is followed by enzymatic or chemical method to remove contaminating proteins, RNA and other macromolecules. The rigid cell wall of the plant cell is weakened by EDTA in the Solution I. Then the cells are subjected to osmotic shock at 65°C to lyse the rigid cell wall of the plant cells. The cellular proteins are denatured by SDS in Solution II. Extraction with phenol denatures the protein, Chloroform removes SDS- Protein/ polysaccharide complexes as white interface between the aqueous and organic phase after centrifugation and isoamyl alcohol prevents foaming. The high molecular weight genomic DNA in aqueous phase is precipitated with 100% ethanol and further the salts are removed by washing with 70% ethanol.

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The protocol contains guanidine-thiocyanate saltbased buffer for efficient cell lysis, protein denaturation and RNase inactivation to keep mRNA intact. Homogenized cells or tissue lysates containing total RNA are incubated with Oligo-dT. mRNA is selectively absorbed on the Oligo-dT cellulose and other impurities are washed away. The mixture containing mRNA and Oligo-dT-Cellulose is placed in to the column. Finally, mRNA is released by adding Elution Buffer in small volume. Sample volume of 100 mg of tissue sample or 10⁶ cells can be used.

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A phage lysate is used to prepare a large quantity of highly purified phage DNA. Host bacteria grown to A650~0.6 are infected with 105-108 phage /ml. Following infection, the cells are shaken vigorously until cell lysis occurs. Remaining labile cells are lysed with chloroform and all debris is removed with a low speed spin. The phage is concentrated by PEG precipitation (solution II). The purified phage capsids are then degraded and the DNA is isolated. 

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DPPH is a free radical and a scavenger for other radicals. It can be either reduced or oxidized. Reactions on the surface of the electrode in the first stage lead to the formation of an anion DPPH- or cation DPPH+. In the solution, it can be oxidized by strong oxidants, such as hydroxyl radicals, or reduced by antioxidants. Its reduction is, usually, based on the acceptance of a hydrogen atom. It may be a multi-stage reaction. In such case, the electron will be first accepted into the DPPH molecule, followed by the proton, or vice versa. These reactions strongly depend on the environment.

Measurements of antioxidative properties are usually based on the reduction of colored DPPH. Changes of the DPPH color are observed photometrically. The Total antioxidant potential (TAP) measure is the decrease of absorption at l=517nm, after the reaction between radicals and sample. These measurements are interfered by both, the tested samples and reduced DPPH.

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The phage assay is originally a virological assay employed to count and measure the infectivity level of the bacteriophages. This assay is the most widely used technique for the isolation of virus and its purification, and to optimize the viral titers. The basis of plaque assay is to measure the ability of a single infectious virus to form a “plaque” on a concurrent monolayer culture cells. A plaque is developed as a part of infection of one cell by a single virus particle that is followed by the replication of that virus, and finally, the death of the cell. The newly replicated virus particles will later infect and then kill surrounding cells. The procedure requires the use of a Double-Layer Agar (DLA) technique also known as double agar overlay method, in which the hard agar serves 3 as a base layer (to form gel), and a mixture of few phage particles (diluted stock) and a very large number of host cells in a soft agar forms the upper overlay. When the plates are incubated, susceptible Escherichia coli cells multiply rapidly and produce a lawn of confluent growth on the medium. When one phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and release new phage particles which infect other bacteria in the vicinity of the initial host cell. The growth or spread of the new viruses is then restricted or limited to the neighbouring cells by the gel. This cycle is repeated until large numbers of bacteria have been destroyed. The destroyed cells produce single circular, non turbid areas called plaques in the bacterial lawn, where there is no growth of bacteria because the phage progeny originating from single virus particles have multiplied sufficiently to kill bacteria over an easily visible area. Eventually the plaque becomes too large to be visible to our naked eye. Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a plaqueforming unit (PFU) and is used to quantitate the number of infective phage particles in the culture. The number of phage particles contained in the original stock phage culture is determined by counting the number of plaques formed on the seeded agar plate and multiplying this by the dilution factor. For a valid phage count, the number of plaques per plate should not exceed 300 nor be less than 30. Plates showing greater than 300 PFUs are too numerous to count (TNTC); plates showing fewer than 30 PFUs are too few to count (TFTC). The main difference between Plaque Assay and Colony-Counting is that, to count bacteria, we spread about 100 - 400 bacteria over the surface of the agar and incubate the plate. To count phage, we spread 100 - 400 phage particles mixed with a very large number of host bacterial cells over the surface of the agar and incubate the plate. In colony-counting, we directly observe the accumulation of large numbers of bacteria, each pile being a colony. In plaque formation, we directly observe the absence of bacteria against a background of dense bacterial growth, each zone of destruction being a phage plaque (hole) in the lawn of bacteria. Each hole represents one phage in the sample applied to the plate.

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This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (eg. Isopropanol, DMSO) and the released, solubilized formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells.

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Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. Removal of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology). DNA is usually recovered by precipitation using ethanol or isopropanol.

The choice of a method depends on many factors: the required quantity and molecular weight of the DNA, the purity required for downstream applications, and the time and expense. Home-made methods often work well for researchers who have developed and regularly use them. However, they usually lack standardization and therefore yields and quality are not always reproducible. Reproducibility is also affected when the method is used by different researchers, or with different sample types.

The separation of DNA from cellular components can be divided into four stages:

1.     Disruption

2.     Lysis

3.     Removal of proteins and contaminants

4.     Recovery of DNA

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                     This kit is designed for fast isolation of genomic DNA from plant source. The kit contains a membrane embedded in a spin column for binding up to 10ug of genomic DNA. Nucleotides, proteins, salts, and other impurities do not bind to the Column. Purified genomic DNA can be applied in most molecular biology experiments including restriction digestion, PCR, Southern-blotting Finger printing etc.

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              A silica-gel membrane that selectively absorbs up to 10mg of DNA fragments in the presence of specialized binding buffers. Nucleotides, oligos, enzymes, mineral oil and other impurities do not bind to the membrane and are washed away. The DNA fragments can then be eluted off the column in small volume and used in almost any downstream applications.

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DTNB reacts with a free sulfhydryl group to yield a mixed disulfi de and 2-nitro-5-thiobenzoic acid (TNB; see Figure target of DTNB in this reaction is the conjugate base (R—S-) of a free sulfhydryl group. TNB is the “colored” species produced in this reaction and has a high molar extinction coeffi cient in the visible range. The molar extinction coeffi cient of TNB is reported to be 13,600 M-1cm-1 at 412 nm and pH 8.0.

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Bacterial protein extraction kit is designed for the lysis of bacterial cells for the purification of recombinant type proteins. It is useful for the preparation of high inclusion bodies. This method does not require special equipment’s such as sonicater or French press.

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Many different methods and technologies are available for the isolation of chromosomal DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA. Removal of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology). DNA is usually recovered by precipitation using ethanol or isopropanol.

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