DNA LAB 5 PDF - Biochemical Techniques

Lab - 1 Biochemical techniques DNA Structure DNA is a molecule that carries most of the genetic instructions used in the development, functioning and reproduction of all known living organisms and ma...

Lab - 1 Biochemical techniques DNA Structure DNA is a molecule that carries most of the genetic instructions used in the development, functioning and reproduction of all known living organisms and many viruses. However, a number of viruses use RNA (ribonucleic acid) as the building block for their genome. DNA and RNA are polymeric molecules made up of linear chains of subunits called nucleotides. Each nucleotide has three parts: a nitrogenous base, a five-carbon-atom sugar and a phosphate group. The combination of base and sugar is termed a nucleoside, while the base–sugar–phosphate is called a nucleotide. Since they contain the sugar 2-deoxyribose, the nucleotides of DNA are termed deoxyribonucleotides, while those of RNA, which contain the sugar ribose, are known as ribonucleotides. The nucleotide bases can be either a double-ringed purine or a single- ringed pyrimidine. DNA and RNA are both built up from two purine containing nucleotides and two pyrimidine containing nucleotides. The purines of both DNA and RNA are the same adenine (A) and guanine (G). The pyrimidine cytosine (C) is also found in both nucleic acids, while the pyrimidine thymine (T) is limited to DNA, being replaced by uracil (U) in RNA. Lab - 1 Biochemical techniques Lab - 1 Biochemical techniques Individual nucleotides are connected to each other in both DNA and RNA through sugar–phosphate bonds that connect the hydroxyl group on the 3-carbon of one nucleotide with the phosphate group on the 5- carbon of another nucleotide by a covalent bonds called phosphodiester bond. Two nucleotides connected to each other are called a dinucleotide, three are called a trinucleotide and numerous nucleotides connected in a long chain is termed a polynucleotide. In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart, either by a mechanical force or high temperature. Lab - 1 Biochemical techniques Buffer preparation Concentrations of a diluted reagent: By using the following formula C1V1 = C2V2. ex: prepare 10 mL with 15% concentration of fructose if you have a stock solution with 50% concentration? Solution: C1V1 = C2V2 50 X V1 = 15 X 10 V1= 150/50 = 3 mL so, we take 3 mL from stock and complete the volume by 7mL of distilled water to reach the final volume. note: you should be sure that all the units are the same. Diluting Molar Solutions: by using M1V1=M2V2 ex: if you have 5M TES buffer, how can you prepare 250mL with 2M? Solution: M1V1=M2V2 5 X V1= 2 X 250 V1= 500/5= 100mL Molarity: Wt 1000 Molarity = ----------- × ---------- M.wt V Lab - 1 Biochemical techniques ex: prepare 3M of NaoH in 400mL ? Solution: M.wt for NaoH can be calculated as below: atomic weight for Na = 23, so, 23*1=23, because we have only one molecule of Na. Atomic weight for oxygen O= 16, so, 16*1=16. Atomic weight for hydrogen H =1, so, 1*1=1 Finally the M.wt is 23+16+1= 40 Wt 1000 Molarity = ----------- × ---------- MW V Wt 1000 3 = ----------- × ---------- = 48 g 40 400