Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 22

11. Hold the beaker at a 45° angle and spool the bacterial DNA out of the solution. Slowly rotate a stirring rod clockwise, as well as up and down, and around, through the layers. Rotate and scoop at the interface instead of swirling. Every once in a while, pull up the rod and examine the DNA strands. 12. Try to spool all of the DNA strands. Observe and record the characteristics of the DNA spooled sample in the data table. 13. Blot the excess EtOH off the sample and place the DNA into a sterile, capped, conical centrifuge tube containing 2 mL of TE buffer. Immediately, observe and record the characteristics of the DNA sample in the data table. 14. Allow the DNA to go back into solution, over several days to a week, before using it for further analysis. After a week, record the sample’s appearance in the data table. Store the samples at 4°C for 2 to 3 weeks. Long-term storage of the DNA samples should be at –20°C (see Figure 4.16). 15. Test the sample for DNA using EtBr testing, G-Biosciences’ NUCLEIC dotMETRIC™ kit or by running a gel (Lab 4j or Lab 4k). Data Analysis/Conclusion Describe the quality and quantity of DNA extracted from the bacterial cell sample compared with other DNA samples you have spooled. Discuss how effective the DNA extraction technique is at isolating pure DNA. Give evidence for your statements. Propose variations in the protocol that may lead to improved quantity or purity of the DNA sample. Thinking Like a Biotechnician 1. Protease is used in this experiment to chop up protein contaminants. There are many different kinds of proteases. One protease that can be purchased at the grocery store is papain, a protease derived from papayas, which is found in meat tenderizers, such as Adolph’s® meat tenderizer (by Lawry’s Foods, Inc.). How can one know that 1 mg/mL of papain is the best concentration of the protease to use? Describe a simple experiment to determine the best concentration for protease activity. 2. You used 10% SDS in the experiment to explode the bacteria cells and precipitate protein contaminants. One can purchase 20% SDS commercially. How much 20% SDS would you need to have enough to make 2000 mL of 10% SDS? 3. The genomic DNA that was spooled was considerably less in volume than the salmon sperm DNA spooled in a previous lab experiment. What is the reason for the difference in DNA yield? DNA Isolation and Analysis 87