Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 21

Materials E. coli broth cultures (from Lab 4g) Pipets, 10 mL Pipet pump, green Tubes, 15-mL, conical Tube racks for 15-mL tubes SDS, 10% Water bath, 65°C RNase, 0.1 mg/mL Protease, 0.1 mg/mL 5 M NaCl (from Lab 4a) Centrifuge for 15-mL tubes Beakers, 50 mL Ethanol (EtOH), 95% Glass rods TE buffer (from Lab 4a) Pipets, 2 mL Pipet pump, blue Procedure Safety Precautions • Do all work in a sterile laminar flow hood, biosafety cabinet hood or on a disinfected countertop. • Use all standard precautions with the Bunsen burner, such as tying back hair, wearing goggles, etc. • Dispose of any bacteria-contaminated products in autoclave bags and/or 10% bleach solution. 1. Using sterile technique, add 10 mL of E. coli broth suspension to a 15-mL capped, conical, centrifuge tube. 2. Add 0.5 mL of 10% SDS to the tube with E. coli. Invert gently five times (5X) to mix. 3. Incubate tube in a 65°C water bath for 15 minutes. 4. Cool on ice for 5 minutes. 5. If desired, add 0.5 mL of RNase and 0.5 mL of protease to the tube. Invert to mix. 6. Add 0.5 mL of 5 M NaCl. Place on ice for 5 minutes. 7. Spin the tube in a tabletop centrifuge for 10 minutes (see Figure 4.15). 8. Gently pipet the supernatant (top layer) to a clean, cold, 50-mL beaker without taking up the loose precipitant of cellular waste. Observe the color and viscosity of the solution. Create a data table in your notebook to record these and other observations. 9. Place the beaker containing supernatant on ice for 5 minutes. 10. Layer 5 mL of ice cold 95% ethanol slowly, with a pipet, down the inside of the beaker. Look at the interface between the alcohol layer and the DNA layer. Do you see any evidence of DNA? Observe the color and viscosity of the solutions and interface. Record these observations into the data table. Note: Before spooling, add 2 mL of TE buffer to a 15-mL conical tube for use in Step 13. mixture before centrifuging supernatant precipitant Heavy sample is pulled down and to the side. Substances are pulled down based on mass. Figure 4.15. Supernatant/precipitant centrifuge results. Figure 4.16.  DNA, RNA, and most proteins are temperaturesensitive and may be degraded or denatured by enzyme contaminants (eg, proteases or DNases) at room temperature. To decrease their activity and preserve molecular or cellular samples, most are stored at –20°C or –8 0