Hana Hakami 2011 DNA Extraction Introduction Why do people want to obtain genomic DNA? There are a number of reasons. For example, this DNA could be used to clone new genes or look for special regions of interest. You could also obtain a number of different genomic DNA and make comparisons with them to identify genetic diseases. The first step in obtaining DNA involves breaking open the cell’s membrane by using physical or chemical means. For example, you could use ultrasonic waves or vibrations (sonification). Alternatively, our method involves homogenization using a mortal and pestle and detergent to disrupt the cells’ membrane. Procedure for DNA extraction DNA will be extracted from whole blood using Traditional Method (John M. S. Bartlett and Anne White). There are many differing protocols and a large number of commercially available kits used for the extraction of DNA from whole blood. This procedure is one is used routinely in both research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tissues or cell cultures (omitting the red blood lysis step. Principle Most mammalian whole blood and bone marrow specimens contain both non-nucleated cells (red blood cells) and nucleated cells (white blood cells) which contain DNA. When purifying DNA from these whole blood or bone marrow specimens, the red blood cells, which lack genomic DNA, are first lysed to facilitate their separation from the white blood cells. Hana Hakami 2011 DNA is purified from cells such as white blood cells, animal tissue, cells contained in body fluids, or microbes, by first lysing the cells with an anionic detergent in the presence of a DNA stabilizer. The DNA stabilizer works by limiting the activity of DNases that are contained in the cell and elsewhere in the environment. Contaminating RNA is then removed by treatment with an RNA digesting enzyme. Other contaminants, such as proteins, are removed by salt precipitation. Finally, the genomic DNA is recovered by precipitation with alcohol and dissolved in a buffered solution containing a DNA stabilizer. Materials Glass wares - 15 ml Centrifuge tubes - 1.5 ml Microfuge tubes - Pasteur pipettes - Tissues - Gloves – Biohazard Bags & Container Equipments Centrifuge – Waterbath set at 65⁰C - roller/rotator - Vortex – pH meter - Freezer (-20⁰C) – Sensitive Balance – Ice Maker Chemicals Tris base - EDTA - Sodium Per-chlorate - Sucrose - MgCl2 - NaCl - Chloroform - Ethanol - SDS (Sodium Dodecyl Sulphate) - Trition x-100 - HCl - NaOH - Clorox Reagents 1. Tris-HCl (1 M) pH7.6 : 121.1g Tris base - 800 ml d.H2O Adjustment pH to 7.6 by HCl. 2. EDTA (0.5 M) pH 8.0 : 146.1 g EDTA - 800 ml d.H2O Adjustment pH to 8.0 by NaOH Make up to 1 L d.H2O. 3. Sodium Per-chlorate (5 M) : 70 g Sodium Per-chlorate - 80 ml d.H2O Make up to 100 ml d.H2O. *You can prepare 5 M Sodium Perhclorate by use Sodium Chloride (29.250g NaCl wet in 100 ml d.H2O). Hana Hakami 2011 4. Reagent A (RBC Lysis Solution): 10 ml 1M Tris-HCl (pH 7.6)– 109.54 g Sucrose - 0.47 g MgCl2 - 10 ml Trition x-100 Make up to 800 ml d.H2O Adjustment pH to 8.0 Make up to 1 L d.H2O. 5. Reagent b (Cell Lysis Solution) : 400 ml 1M Tris-HCl (pH 7.6)/ 120 ml 0.5 M EDTA (pH 8.0) - 8.76 g NaCl Adjustment pH to 8.0 Make up to 1 L d.H2O 10 g SDS. 6. TE Buffer pH 7.6 (DNA Hydration Solution): 10 ml 1M Tris-HCl (pH 7.6) - 2ml 0.5 M EDTA (pH 8.0) Make up to 1 L d.H2O Adjustment pH to 7.6. Protocol 1st: RBC Lysis 1. Prepare 3 ml of whole blood sample in EDTA tube. 2. Transfer the blood sample to 15 ml tube. 3. Add 12 ml Reagent A. 4. Mix on rolling for 4 min at room temperature. 5. Centrifuge at 3000 g for 5 min at room temperature. 6. Discard supernatant gently . 7. Remove moisture by inverting the tube on tissue paper. 2nd: Cell Lysis 1. Add 1 ml Reagent B on cell pellet. 2. Vortex briefly to re-suspend the cell pellet. 3. Add 250 ml 5 M sodium per-chlorate. 4. Mix by inverting tube several times. 5. Place tube in water bath for 15- 20 min at 65⁰C. 6. Cool to room temp. 3rd: Protein Precipitation 1. Add 2 ml ice-cold chloroform. 2. Mix on a rolling or rotating mixer for 30-60 min. 3. Centrifuge at 2500g for 5 min. 4. Transfer upper phase into a clean 15 ml tube using a sterile pipette. Hana Hakami 2011 4th: DNA Precipitation 1. Add 2 to 3 ml ice- cold ethanol gently on tube wall and invert gently to allow DNA to precipitate. 2. Centrifuge at 2500g for 3 min. 3. Discard from supernatant gently. 4. Allow to air dry the DNA pellet by invert the tube on absorption tissue for 10-20 min . 5th: DNA Hydration 1. Re-suspend the pellet in 100-300 µl of TE buffer. 2. Rehydrate DNA by incubating at 65°C for 1 hour and leave it overnight at room temperature. 3. Centrifuge the DNA sample briefly by short spin (Spin-down). 4. Transfer the DNA sample to 1.5-ml Eppendorf tube (microfuge tube) * Do not forget fill full information about DNA sample on Eppendorf tube (name, type of sample and date ). * As a final step in nucleic acid isolation, the yield and purity of the extracted nucleic acid may need to be determined. 6th: Storage of DNA Original stock of each sample of DNA will be labeled and stored at -80°C. From each sample 100 µl working DNA (for example; 50ng/µl for all working DNA samples) will be labeled and stored at 4°C or -20° C.
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