Biotech 2nd Edition Sample Ch. 4 Biotechnology Sample Ch. 4 - Page 31

Fill in the reaction matrix below to show how each was prepared. Think about how you will make these. “1:10” means 1 part sample in a total of 10 parts (sample and solvent). How much of which sample and how much diluent do you need in each tube? Diagram how you will make the 3 samples. Tube # Dilution from the 0.5 µg/µL stock 1 Undiluted 2 3 4 1:10 1:100 1:1000 Amount of Which Lambda DNA Sample is added to the Tube? 22 µL, 0.5 µg/µL stock Amount of dH2O added as diluent to the Tube? Amount of DNA in tube after preparation of all samples? 0 µL 10 µg 4. Prepare a 5th tube with only 20µL of dH2O to use as a negative control and two tubes of 20µL of unknown sample (one undiluted and one diluted 1:2). 5. To each of the five known DNA sample tubes and 2 unknown sample tubes, add either 10X DNA loading dye (for TAE gels) or 5X LB loading dye (for LB gels). How much of the concentrated loading dye are you adding to each tube? Pipet up and down to mix the sample with the loading dye. Change tips each time you add loading dye to a new tube. 6. Pour 1X electrophoresis buffer over the gel (2 mm over the entire gel). Gently pull the comb up and out to reveal the wells. 7. Into the first row, load all of each of your known standard samples into the wells of the gel, loading Wells 1–5, from highest concentration standard to lowest. Into Well 6 add 10µL of DNA sizing standard prepared by the supervisor. Change tips with each load. 8. Into Wells 3 and 5 of the 2nd rows, add all of the undiluted and diluted 1:2 unknown samples, respectively. 9. Close the gel box and run the electrophoresis at the recommended voltage for the type of electrophoresis buffer used in the gel and gel box (either 110V/60mA for TAE gels or 300V/80mA for LB gels). Record the gel run conditions. 10. Once the gel run is completed, turn of the gel box, gently slide the gel into a large, labeled weigh boat and stain it by the method recommended by your supervisor. 11. Observe and photograph the stained gel and observe the amount of staining for each concentration (and mass) of DNA in the known standards. Study the unknown sample bands. Compare the amount of staining in these as compared to the unknowns. Estimate the concentration (and mass) of DNA of the unknown samples in the gel using the standard known samples as a comparison. Data Analysis and Conclusion Report the estimated concentration (µg/µL) of the unknown (undiluted and diluted) samples. Describe how you determined these values. Knowing that 20µL of each unknown sample was used in the gel, what is the mass of DNA (µg) in these samples? Discuss how well your standards shows the range of DNA amounts? Were any of the samples so dilute that the DNA was not visible? List technical errors in setting up the experiment that might lead to erroneous data. Discuss the value of learning how to quantify DNA by this method. How would this technique be valuable to biotechnologists? Thinking Like a Biotechnician 1. The original stock unknown sample contained a volume of 500 µL. Based on your estimation of the mass of DNA in the unknowns you ran on the gel, what is the total mass of Lambda DNA in the stock unknown sample? 2. Is it possible to estimate the sizes (bp) of the Lambda DNA pieces in the concentration standards by comparing them to the Lambda/HindIII sizing standards? Which known concentration of the Lambda DNA known samples shows a sharp enough resolution to be able to best determine the size of these DNA pieces? 96 Chapter 4   Laboratory Manual