EXPERIMENT 1: Isolation of Chromosomal DNA from Bacteria TE buffer It is a buffer for storage of DNA & RNA. Thus, its commonly used as buffer solut ion in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule che lating cations like Mg2+ or can be say as an interchelating agent, thus it basic ally takes up free ions form the solution, the reason for this is that most nucl eases make use of ions to catalyse their nuclease ability. The purpose of TE buf fer is to protect DNA or RNA from degradation. SDS SDS Solution (10% w/v) is sodium dodecyl sulphate in distilled, deionized water. SDS is an anionic detergent which disrupts cell membrane and destabilizes all h ydrophobic interactions holding macromolecules in their native form. It is used in polyacrylamide gel electrophoresis for the determination of protein molecular weight according to their electrophoretic mobility (a function of length of pol ypeptide chain or molecular weight). Proteinase K Proteinase K is a broad-spectrum serine protease. Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aro matic amino acids with blocked alpha amino groups. It is commonly used for its b road specificity. Proteinase K is commonly used to digest protein and remove con tamination from preparations of nucleic acid. Addition of proteinase K to nuclei c acid preparations rapidly inactivates nucleases that might otherwise degrade t he DNA or RNA during purification. It is highly-suited to this application since the enzyme is active in the presence of chemicals that denature proteins, such as SDS and urea, chelating agents such as EDTA, sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors. Proteinase K is also stable over a wide pH r ange (4-12), with a pH optimum of pH 7.5-12.

Phenol-Chloroform The word phenol is also used to refer to any compound that contains a six-member ed aromatic ring, bonded directly to a hydroxyl group (-OH. Its chemical name is hydroxybenzene. This is particularly important if the phenol is mixed with chlo roform (a commonly-used mixture in molecular biology for DNA & RNA purification from proteins). Extraction with phenol and phenol/chloroform mixtures is a unive rsal method for purification of DNA and RNA. Phenol used to break down the prote in in the cell wall it is because the remaining proteins will denature and preci pitate for the most part in the phenol. This will form a white precipitate at th e interface between the phenol and the water. The chloroform in disrupting prote in secondary structure causing proteins to denature and precipitate from solutio n. Although each of these solvents is capable of performing this function alone, the two materials together remove proteins from solution much more effectively. Nucleic acids are recovered in the liquid phase. During phenol extraction, the precipitated proteins collect at an interphase between the aqueous and organic l ayers. It is very important to understand the effect of pH on the performance of phenol. For the purification of DNA, the use of phenol at pH above 7.0 is desir able in order to collect DNA in the upper aqueous layer of a phenol extraction ( phenol saturated, pH8: UP87336). At lower pH (4.5), DNA and proteins are collect ed at the interphase although RNA can be collected in the aqueous phase (phenol saturated, pH4,5: UP893305). Phenol and chloroform are supplied as solution alon e, premixed with chloroform and isoamyl alcohol to reduce the extraction time. Chloroform

Chloroform is the organic compound with formula CHCl3. This colorless, sweet-sme lling, dense liquid is a trihalomethane. It is also considered somewhat hazardou s. Chloroform is a common solvent in the laboratory because it is relatively unr eactive, miscible with most organic liquids, and conveniently volatile. Chlorofo rm is used to dissolve non-nucleic acid biomolecules in DNA and RNA extractions, its remove proteins from nucleic acid samples. NaCl Sodium chloride, also known as salt, common salt, table salt, or halite, is an i onic compound with the formula NaCl. The function of NaCl for this experiment is to repair of broken DNA. NaCl provides Na+ ions that will block negative charge from phosphates on DNA. Negatively charged phosphates on DNA cause molecules to repel each other. The Na+ ions will form an ionic bond with the negatively char ged phosphates on the DNA, neutralizing the negative charges and allowing the DN A molecules to come together. Ethanol Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alc ohol, is a volatile, flammable, colorless liquid, and its molecular formula is C 2H5OH. Ethanol use for the precipitation of extracted DNA. It causes the DNA to precipitate and come out of the solution. DNA will rise into the alcohol layer.

EXPERIMENT 2: Plasmid DNA Isolation (miniprep) Reagent: LB medium Lysogeny broth (LB), a nutritionally rich medium, is primarily used for the grow th of bacteria. It is also known as Luria broth or Luria-Bertani broth. These me dia have been widely used in molecular microbiology applications for the prepara tion of plasmid DNA and recombinant proteins. Solution I (TRIS, EDTA, glucose, distilled water) TRIS Tris (also known as THAM) is an abbreviation of the organic compound known as tr is(hydroxymethyl) aminomethane, with the formula (HOCH2)3CNH2. Tris buffer (dilu ted buffer concentrate for RNase) is used to resuspend the DNA precipitate in a higher concentration. The buffer contains the enzyme RNase, which further degrad es RNA. EDTA EDTA is widely used initialism for the chemical compound ethylenediaminetetraac

etic acid. The conjugate base is named ethylenediaminetetraacetate. EDTA is a po lyamino carboxylic acid and a colourless, water-soluble solid. EDTA is added to some buffer solutions to remove divalent cations and prevent microbial growth. E DTA binds divalent metal ions (Ca2+, Mg2+, Mn2+) that could form salt with anio nic PO43-group of DNA. It also destabilizes the cell membrane and prevents preci pitation of DNA. EDTA also inhibits DNAases. GLUCOSE Glucose (Glc) is a monosaccharide (or simple sugar) is a very important carbohy drate in biology. The glucose is used in this experiment to stabilize the osmoti c pressure. DISTILLED WATER Distilled water is water that has virtually all of its impurities removed throug h distillation. Distillation involves boiling the water and then condensing the steam into a clean container, leaving most if not all solid contaminants behind . Distilled water act as a solvent in this experiment. Solution II (NaOH, SDS, distilled water) NaOH NaOH, also known as lye and caustic soda, is a caustic metallic base. Sodium hy droxide is a common base in chemical laboratories. The NaOH. It also tends to de natures macromolecules by changing the condition of ionizable groups (ionizing certain groups and deionizi ng others). SDS SDS Solution (10% w/v) is sodium dodecyl sulphate in distilled, deionized water. SDS is an anionic detergent which disrupts cell membrane and destabilizes all h ydrophobic interactions holding macromolecules in their native conformation. It is used in polyacrylamide gel electrophoresis for the determination of protein m olecular weight according to their electrophoretic mobility (a function of lengt h of polypeptide chain or molecular weight). Solution III (potassium acetate, glacial acetic acid, distilled water) POTASSIUM ACETATE. The low pH of the Potassium acetate solution neutralizes the NaOH and precipitat es the SDS from solution, along with the cellular debris.when the pH returns to near-neutrality then the macromolules renature. POTASSIUM ACETATE The potassium acetate neutralization buffer contains acetic acid and potassium salts. The acidic buffer neutralizes the alkaline conditions created by the sodium hydroxide. This is really the key step in the alkaline lys is procedure. The low pH of the potassium acetate solution neutralizes the NaOH and when the p H returns to near-neutrality then the macromolecules renature. At the same time the potassium ions react with the dodecyl sulfate to produce an insoluble materi al which no longer keeps the macromolecules in solution.

GLACIAL ACETIC ACID Glacial acetic acid is an excellent polar protic solvent, as noted above. It is frequently used as a solvent for recrystallization to purify organic compounds. Pure acetic acid is used as a solvent in the production of terephthalic acid (TP A), the raw material for polyethylene terephthalate (PET). The acetic acid neutr alizes the pH, allowing the DNA strands to renature. PHENOL Phenol, also known as carbolic acid, is a toxic, white with a slightly pink ting

e, crystalline solid. Its chemical formula is C6H5OH and its structure is that o f a hydroxyl group (-OH) bonded to a phenyl ring, making it an aromatic compound . Phenols effective for breaking down protienacious cellular walls or viral caps ids.for this experiment, phenol used to break down the protein in the cell wall it is because the remaining proteins will denature and precipitate for the most part in the phenol. CHLOROFORM Chloroform is the organic compound with formula CHCl3. This colorless, sweet-sme lling, dense liquid is a trihalomethane. Chloroform is used to dissolve non-nuc leic acid biomolecules in DNA and RNA extractions and remove proteins from nucl eic acid samples. It is used for further denatures and coagulates the protein, so that they collect at interface between the aqueous the organic phase upon cen trifugation. ABSOLUTE ETHANOL Alcohol is not intended for human consumption. It may contain trace amounts of t oxic benzene if azeotropic distillation is used to remove water. Absolute ethano l is used as a solvent for laboratory. Absolute ethanol evaporates much faster t han a solution of 50% isopropanol. This "ethanol wash" will speed up the process because the next step is to evaporate the alcohol used in the precipitation ste p. 70% ETHANOL 70% Ethanol is to remove any residual salt. TE BUFFER Its commonly used as buffer solution in molecular biology, especially in proced ures involving DNA or RNA. "TE" is derived from its components: Tris, a common p H buffer, and EDTA, a molecule chelating cations like Mg2+ or can be say as an i nterchelating agent, thus it basically takes up free ions form the solution, the reason for this is that most nucleases make use of ions to catalyse their nucle ase ability. The purpose of TE buffer is to protect DNA or RNA from degradation. TE buffer is commonly used to redissolve DNA because it contains EDTA. The EDTA will chelate magnesium ions, which are a cofactor for most nucleases (enzymes w hich degrade nucleic acids). If the DNA prep becomes contaminated with a nuclease (l ike the ones produced by the cells in skin) then the nuclease will be inactivate dby the fact that the magnesium cofactor is unavailable in the solution (because it is chelated by the EDTA).