Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 2

Laboratory 4a Making Solutions for DNA Isolation Background For genetic engineering, PCR, or other work with DNA, a pure DNA sample is required. DNA must be purified from cells, removing all other cellular constituents and contaminant molecules. Many purification protocols include a step to remove protein contaminants using a salt solution. One of the final steps in DNA isolation is to precipitate DNA, or take the DNA out of solution. In most cases, DNA precipitation is done using alcohol. In several upcoming laboratory activities, DNA strands will be precipitated from solution. In Laboratory 4b, genomic salmon testes DNA will be precipitated onto a glass rod. To increase the number of DNA strands that will spool around the glass rod, 5 M sodium chloride (NaCl) is added to the solution prior to alcohol precipitation. The Na+ ions in a NaCl solution bind to the DNA, decreasing its negative charge, allowing DNA molecules to come closer together and spool more easily. Isolated DNA can be stored for long periods in sterile TE buffer (containing TRIS and EDTA). The TE buffer contains TRIS to maintain the pH of the DNA sample and EDTA to denature any DNases, which might contaminate the sample. In this activity, you will prepare 5 M NaCl solution and TE buffer. Purpose To make 10 mL of 5 M NaCl solution. To make 100 mL of TE buffer (10 mM TRIS, 1 mM EDTA, pH 8.0) Materials Balance, analytical Balance, tabletop milligram Weigh paper, 7.6×7.6 cm Weigh boat, 3.5”×3.5” Lab scoops Sodium chloride Tubes, 15 mL, conical Tube racks for 15-mL tubes TRIS EDTA, disodium salt Media bottle and cap, 125 mL pH paper, wide/narrow-range Hydrochloric acid Sodium hydroxide Glass rods Procedure Part I: Preparation of 5 M of NaCl 1. Determine the mass of NaCl to be measured. Remember, you want enough NaCl to give a concentration of 5 M, but you only want to make 10 mL of this solution. In your notebook show the calculations and draw a diagram of how the solution will be prepared. 2. Weigh the amount of NaCl needed and place it in a 15-mL conical tube. Slowly add dH2O, while stirring, until a final volume of 10 mL is reached. 3. Cap the tube and invert it for several minutes until the salt dissolves completely into solution. Check to make sure that the volume is still 10 mL. If it is not, add a tiny amount of dH2O until the final volume is 10 mL. Label the tube with the sample name, concentration, date, and technician’s initials. Store at 4°C until ready to use. Part II: Preparation of TE Buffer Note: The calculations for each solute (TRIS and EDTA) are done separately based on a final volume of 100 mL. 1. Determine the mass of TRIS to be measured (from the bottles in the chemical storeroom) to give the correct concentration and volume in the final TE buffer. Show the calculations in your notebook. 2. Determine the mass of EDTA to be measured (from the bottles in the chemical storeroom) to give the correct concentration and volume in the final TE buffer. In your notebook show the calculations and draw a diagram of how the TE buffer solution will be prepared. DNA Isolation and Analysis 67