DNA Isolation by Spooling Method PDF

❖ DNA, organic chemical of complex molecular structure that is found in all prokaryotic and eukaryotic cells and in many viruses. ❖ DNA codes genetic information for the transmission of inherited traits. ❖ Each strand of a DNA molecule is composed of a long chain of monomer nucleotides. ❖ T...

❖ DNA, organic chemical of complex molecular structure that is found in all prokaryotic and eukaryotic cells and in many viruses. ❖ DNA codes genetic information for the transmission of inherited traits. ❖ Each strand of a DNA molecule is composed of a long chain of monomer nucleotides. ❖ The nucleotides of DNA consist of a deoxyribose sugar molecule to which is attached a phosphate group and one of four nitrogenous bases: ❖ Two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). ❖ The nucleotides are joined together by covalent bonds between the phosphate of one nucleotide and the sugar of the next, forming a phosphate-sugar backbone from which the nitrogenous bases protrude. ❖ One strand is held to another by hydrogen bonds between the bases; i.e., adenine bonds with thymine, and cytosine with guanine. ❖ The two strands are antiparallel. ❖ Structure of DNA helix was discovered by James Watson and Francis Crick. ❖ Escherichia coli (E. coli) is a bacteria that is commonly found in the lower intestine of warm-blooded organisms, facultative anaerobe. ❖ It is a gram negative bacteria, because its cell wall is composed of a thin peptidoglycan layer and an outer membrane. ❖ It picks up the color of the counterstain safranin and stains pink ❖ Outer membrane surrounding the cell wall provides a barrier to certain antibiotics. ❖ Most strains are harmless, but some can cause serious food poisoning. 1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml microcentrifuge tube and centrifuge at max speed for 1min to pellet the cells. 2. Discard the supernatant. Note: Remove as much of the supernatant as you can without disturbing the cell pellet. 3. Resuspend the cell pellet in 200 μl GTE mix buffer and tap to completely resuspend cell pellet. 4. Add 400 µl of lysis buffer conataining 1% SDS to the re-suspended cells. 5. Invert mix gently and incubate for 2 minutes at room temperature. 6. Add an equal volume of chilled isopropanol to precipitate DNA. 7. Mix well by inverting the tube until the goopy mucus transparent white threads observe (DNA). 8. The DNA can be scooped up and swirled around a glass rod or wooden spooling stick. This is called DNA spooling.  Chemicals 1. Luria Broth medium to grow E. coli: Tryptone - 1.0g Yeast extract - 0.5g NaCl - 0.5g Glucose - 0.1g. Dissolve the components in 75ml of distilled water, adjust the pH to 7.6 and finally make the volume to 100ml. Sterilize by autoclaving. Luria Broth Glucose Tris-HCl 2. GTE mix: 50mM Glucose 50mM Tris-HCl 10mM EDTA. Sterilize by autoclaving. 3. 10% SDS: Dissolve 10g of SDS in 80 ml of distilled water adjust pH to 7.2. Raise the volume to 100ml. Do not Autoclave. EDTA SDS Isopropanol (Chilled) 4. 70% Ethanol ❖ Nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired. ❖ In GTE mix, ❖ Glucose: Maintain the osmolarity. ❖ Tris-Cl: Maintain the pH, preventing acid hydrolysis of DNA. ❖ EDTA: DNA is to be protected from endogenous nucleases by chelating Mg2+ ions using EDTA. Mg2+ ion is a necessary cofactor for action of most of the nucleases. ❖ SDS: Cells are broken using a lysis buffer (mostly a detergent). ❖ Nucleoprotein interactions are disrupted with SDS, phenol or proteinase K. ❖ Alcohol or Isopropanol: DNA precipitation. ❖ Macromolecules are inactivated using enzymes such as proteases and RNases. ❖ The genomic DNA is usually visible as a gelatinous, white mass and can be stored frozen at –80°C for several years. THANK YOU