DNA Extraction From Fresh Bone

Extract nucleic acids, such as DNA, from bone samples in order to analyze gene expressions, to look for somatic mutations of tumors or other pathological tissue, or for genotyping archive material when other sources of DNA are not available. You can use several kits that have already provided by biotech companies. But, if you are extracting DNA from large number samples, you can use a homemade method as described here to be effective in cost. There are four procedures that ascertain the successful extraction of nucleic acids from tissue: 1. disrupting the tissue so that extraction reagents can reach the cells. 2. disrupting the cell membranes so that nucleic acids are liberated. 3. separation of the nucleic acid from other cellular components. 4. precipitation and solubilization of the nucleic acid. Materials 1. DNA extraction buffer: Add 17.6 mL of 0.75 M sodium citrate, pH 7.0, 26.4 mL of 10% sodium lauryl sarkosyl, and 250 g of guanidinium isothiocyanate to 293 mL of distilled water and mix well. Add 7.2 microliter of beta-mercaptoethanol/mL of lysis buffer on the day of use All chemicals should be of molecular biology grade. The solutions can be stored at 4oC for up to 3 months. 2. 0.5 M ETDA: Add 93.05 g of EDTA to 300 mL of distilled water and add 10 N NaOH to pH 8.0. Make up to 500 mL. Autoclave. Tris–EDTA: Add 1 mL of 1 M Tris to 200 microliters of 0.5 M EDTA. Make up to 100 mL with distilled water. 3. 3 M Sodium acetate, pH 5.2: Add 401.8 g of sodium acetate to 800 mL of distilled water. Adjust pH to 5.1 with glacial acetic acid. Make up to 1 L with distilled water. Autoclave. 4. General Reagents: Tris-saturated phenol pH 7.8–8.0 (Sigma), Chloroform, Isopropanol 100%, Ethanol. Methods 1. Collect the bone sample in a sterile container containing phosphate-buffered saline (PBS) and transport to the laboratory within 1–2 h. If the DNA extraction is not initiated immediately, freeze the sample at –20oC or below for later use. 2. Place the bone tissue in a clean glass Petri dish. Using bone cutters or a strong sharp pair of scissors, isolate a piece of bone measuring about 1 cm3 and transfer to a clean 5-mL bijoux container. 3. Add 1 mL of DNA extraction buffer and homogenize the tissue with the scissors until a slurry solution is obtained. 4. Transfer 500 microliters aliquots of slurry into screw-capped conical-bottomed 1.5-mL Eppendorf tubes. 5. Add one volume of Tris-saturated phenol, followed by one volume of chloroform per tube. Mix well by inverting the tubes a few times or by shaking. Do not vortex, because vortex-mixing causes long strands of DNA to shear. 6. Centrifuge the tubes at 10,000 g for 20 min to separate the phases. 7. Transfer the upper layer to a fresh centrifuge tube (taking note of the volume), being careful not to disturb the milky layer at the interface. Repeat steps 5–7 if the interface is disturbed. 8. Add one volume of ice-cold isopropanol and 0.1 volumes of 3 M sodium acetate to the supernatant. Mix well and allow to stand for 15 min on ice. 9. Centrifuge the tubes at 10,000g for 20 min to pellet the DNA. Orientate the Eppendorf tube so that you can identify where the DNA pellet lies. A pellet should be visible the bottom of the tube. 10. Aspirate and discard the supernatant, taking care not to disturb the pellet. Wash the sample with 1.75 mL of ice-cold ethanol and centrifuge at 10,000g for 5 min. Aspirate and discard the supernatant and then repeat the wash. 11. Dissolve the DNA pellets in 10–50 microliters of water or Tris–EDTA buffer (you can pool DNA from the same sample at this stage) and quantitate by spectrophotometry or with Hoechst 33258. Hoechst 33258 is a DNA-specific dye that can be used to quantitate DNA. 12. Store the sample frozen at –20oC or below.

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Benefits of Ayurvedic Medicines

• By using ayurvedic and herbal medicines you ensure physical and mental health without side effects. The natural ingredients of herbs help bring “arogya” to human body and mind. ("Arogya" means free from diseases). The chemicals used in preparing allopathy medicines have impact on mind as well. One should have allopathy medicine only when it is very necessary. • According to the original texts, the goal of Ayurveda is prevention as well as promotion of the body’s own capacity for maintenance and balance. • Ayurvedic treatment is non-invasive and non-toxic, so it can be used safely as an alternative therapy or alongside conventional therapies. • Ayurvedic physicians claim that their methods can also help stress-related, metabolic, and chronic conditions. • Ayurveda has been used to treat acne, allergies, asthma, anxiety, arthritis, chronic fatigue syndrome, colds, colitis, constipation, depression, diabetes, flu, heart disease, hypertension, immune problems, inflammation, insomnia, nervous disorders, obesity, skin problems, and ulcers. Ayurvedic Terms Explained Dosha: In Ayurvedic philosophy, the five elements combine in pairs to form three dynamic forces or interactions called doshas. It is also known as the governing principles as every living things in nature is characterized by the dosha. Ayurvedic Facial: Purportedly, a "therapeutic skin care experience" that involves the use of "dosha-specific" products and a facial massage focusing on "marma points." Ayurvedic Nutrition (Ayurvedic Diet): Nutritional phase of Ayurveda. It involves eating according to (a) one's "body type" and (b) the "season." The alleged activity of the doshas--three "bodily humors," "dynamic forces," or "spirits that possess"--determines one's "body type." In Ayurveda, "body types" number seven, eight, or ten, and "seasons" traditionally number six. Each two-month season corresponds to a dosha; for example, the two seasons that correspond to the dosha named "Pitta" (see "Raktamoksha") constitute the period of mid-March through mid-July. But some proponents enumerate three seasons: summer (when pitta predominates), autumn, and winter (the season of kapha); or Vata season (fall and winter), Kapha season (spring), and Pitta season (summer). According to Ayurvedic theory, one should lessen one's intake of foods that increase ("aggravate") the ascendant dosha.

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Serial analysis of gene expression

Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University and published in 1995. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGEand the most recent SuperSAGE that enables very precise annotation of existing genes and discovery of new genes within genomes because of an increased tag-length of 25–27 bp. Overview SAGE experiments proceed as follows: 1. Isolate the mRNA of an input sample (e.g. a tumour). 2. Extract a small chunk of sequence from a defined position of each mRNA molecule. 3. Link these small pieces of sequence together to form a long chain (or concatemer). 4. Clone these chains into a vector which can be taken up by bacteria. 5. Sequence these chains using modern high-throughput DNA sequencers. 6. Process this data with a computer to count the small sequence tags. Applications Although SAGE was originally conceived for use in cancer studies, it has been successfully used to describe the transcriptome of other diseases and in a wide variety of organisms. Comparison to DNA microarrays The general goal of the technique is similar to the DNA microarray. However, SAGE is a sequence-based sampling technique. Observations are not based on hybridization, which result in more qualitative, digital values. In addition, the mRNA sequences do not need to be known a priori, so genes or gene variants which are not known can be discovered. Microarray experiments are much cheaper to perform, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise.

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Ayurveda

Ayurveda is a system of traditional medicine native to India, and practiced in other parts of the world as a form of alternative medicine. In Sanskrit, the word Ayurvedacomprises the words āyus, meaning 'life' and veda, meaning 'science'. Evolving throughout its history, Ayurveda remains an influential system of medicine in South Asia. The earliest literature of Ayurveda appeared during the Vedic period in India. The Sushruta Samhita and the Charaka Samhita were influential works on traditional medicine during this era. Ayurvedic practitioners also claim to have identified a number of medicinal preparations and surgical procedures for curing various ailments and diseases. Ayurveda is considered to be a form of complementary and alternative medicine (CAM) within the western world, where several of its methods—such as herbs, massage, and Yoga as exercise or alternative medicine—are applied on their own as a form of CAM treatment. Ayurveda stresses the use of vegetable drugs Fats are used both for consumption and for external use. Hundreds of vegetable drugs are employed, including cardamom and cinnamon. Some animal products may also be used, for example milk, bones, and gallstones etc. Minerals—including sulfur, arsenic, lead, copper sulfate, gold—are also consumed as prescribed.. This practice of adding minerals to herbal medicine is known as Rasa Shastra. In some cases alcohol is used as a narcotic for the patient undergoing an operation. The advent of Islam introduced opium as a narcotic. Both oil and tar are used to stop bleeding. Oils may be used in a number of ways including regular consumption as a part of food, anointing, smearing, head massage, and prescribed application to infected areas. The proper function of channels—tubes that exist within the body and transport fluids from one point to another—is seen as vital, and the lack of healthy channels may lead to disease and insanity. Sushruta identifies that blockages of these channels may lead to rheumatism, epilepsy, paralysis, and convulsions as fluids and channels are diverted from their ideal locations. Sweating is favored as a manner in which to open up the channels and dilute the Doshas causing the blockages and harming a patient—a number of ways to take steam bathing and other steam related cures are recommended so that these toxins are released.

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