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PLEASE KEEP THE LAB CLEAN NO FOOD/DRINKING/GUM IN THE LAB

PLEASE KEEP THE LAB CLEAN NO FOOD/DRINKING/GUM IN THE LAB TURN OFF CELL PHONE (except when used as a calculator). Welcome Bio21 Online Hybrid Section 4058 Fall 2012. Thomas (Tom) Chen. Ph.D. from the University of Florida Postdoc: Colorado State University

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PLEASE KEEP THE LAB CLEAN NO FOOD/DRINKING/GUM IN THE LAB

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  1. PLEASE KEEP THE LAB CLEAN NO FOOD/DRINKING/GUM IN THE LAB TURN OFF CELL PHONE (except when used as a calculator) Welcome Bio21 Online Hybrid Section 4058 Fall 2012

  2. Thomas (Tom) Chen Ph.D. from the University of Florida Postdoc: Colorado State University Fellow: National Institutes of Health Professor: The University of Tennessee, Knoxville Visiting Professor: Medical Univ. of S. Carolina Stanford University My curriculum vitae is posted in Doc Sharing.

  3. Course Home Threaded Discussion Threaded Discussion Grading General Comments/Questions Test 1 Test 2* Test 3* Test 4* Test 5* Test 6* Final Exam* Career Choice/Advice Lab Quiz #1 Lab quiz 2* Library Assignment I Library Assignment 2* Standard Curve Lab Diffusion Lab Report* Enzyme Lab Protocol* Enzyme Lab* General Comments/Questions II* Enzyme Lab* Libr 2* Libr II Due 1014* Course Home Syllabus Letter of Recommendation Threaded Discussio Libr Assgn 1 Assessment Lectures Tech Info McGraw-Hill Connect Biology Library Assignment II

  4. Today Class roll. Add/Drop Syllabus & Course eCompanion website. Assignment Week 1 Standard Curve DNA Lab procedures

  5. The goals of this exercise are to: • Separate the cellular components of a plant cell to obtain plant DNA. • Develop a consistent process for measuring the concentration of a component in solution. • Process data to make a standard curve graph. • Use the standard curve graph to determine the concentration of the unknown DNA solution. • Use collective standard curve data to evaluate the variability that occurs during experimentation. • Write a laboratory report that presents the data and your interpretation of the data.

  6. GOOD LABORATORY PRACTICE • Please read and be familiar with Lab Safety Rules. • Please handle the cuvettes and other lab instruments with care, e.g., do not slam close the sample lid. • Clean up everything that you used on your bench and more importantly, the common area that you have used. • Do not forget to take the cuvette out of the spec when done. • Dispose of wastes in proper containers. • Be careful with every step that you carried out. • Record keeping: Record every observation that you have made.

  7. The exercise is divided into three parts A. Strawberry DNA Extraction B. Standard Curve Preparation C. DNA Quantitation • DNA is enclosed in a nuclear and a cell membrane made of phospholipids • DNA is also coiled around proteins • Both the phospholipid layer and the proteins must be removed to see DNA

  8. To Extract DNA, You Must Remove … • Cell membrane • Cytoplasm • Nuclear membrane • Proteins

  9. strawberry in a zip-loc bag. • 10 ml of DNA Extraction Buffer. • plastic funnel with cheesecloth. Place the funnel in a 150 ml beaker. • plastic transfer pipette. • ice-cold ethanol. • Add 0.5 ml of 70% ethanol to a 1.5 ml microfuge tube. 2x • wooden stick. • 5 drops of 0.2% methylene blue. • tap water. 2x, 5 ml tap water.

  10. A. Strawberry DNA Extraction Set up the cheesecloth funnel first. Place one strawberry in a zip-loc bag and seal the top of the bag completely. Mash or squash the strawberry with your hands for about 2 minutes.

  11. A. Strawberry DNA Extraction Add 10 ml of DNA Extraction Buffer. This buffer contains detergent and salt (sodium chloride). Reseal and mash for 2 more minutes. Line a plastic funnel with cheesecloth. Place the funnel in a 150 ml beaker. Cut a corner from the zip-loc bag and empty the strawberry solution into the cheesecloth. Gently squeeze the solution into the funnel and allow it to collect in the beaker. Minimize foaming

  12. A. Strawberry DNA Extraction Transfer one-third to one-half of the strawberry solution to a 15 ml tube. Using a plastic transfer pipette, slowly add ice-cold ethanol down the side of the tube so that it forms a layer on top of the strawberry solution. DNA dissolves in water, but NOT in alcohol Continue adding the ethanol until the tube is about three-quarters filled and you see the DNA appearing as a white, wispy material at the interface between the strawberry solution and ethanol. Remove the DNA from the tube, being careful not to disturb the interface

  13. A. Strawberry DNA Extraction Add 0.5 ml of 70% ethanol to a 1.5 ml microcentrifuge tube. Place a wooden stick into the tube at the solution-ethanol interface and rotate gently to wind the DNA around the stick. Transfer the DNA from the stick into the 70% ethanol solution. The material will be sticky and you may need to scrape the stick against the edge of the tube or use the transfer pipette to push the DNA from the stick. Use an additional 0.5 ml of 70% ethanol to completely transfer the DNA, ending with a total volume of 1 ml in the microcentrifuge tube.

  14. A. Strawberry DNA Extraction Spin the tube with the DNA DNA forms a pellet on the side of the microcentrifuge tube Pour off the supernatant which is alcohol Let the tube air dry for 5 minutes. Add 5 drops of 0.2% methylene blue to the DNA pellet. Use the wooden stick or a transfer pipette to mix the dye with the DNA. Allow this mixture to incubate at room temperature for 5 minutes.

  15. A. Strawberry DNA Extraction Using a transfer pipette, fill the microcentrifuge tube with tap water. Close the tube and mix the contents by inverting the tube or vigorously tapping the bottom of the tube with your finger. Spin the tube in the microcentrifuge at high speed for 1 minute. Decant as much of the water as possible without disturbing the DNA pellet. Refill the microcentrifuge tube with tap water and repeat the mixing step. Spin the tube in the microcentrifuge at high speed for 1 minute.

  16. A. Strawberry DNA Extraction Remove as much of the water as possible without disturbing the DNA pellet. Using a transfer pipette, resuspend the DNA in 1 ml tap water by taking it up and down in a transfer pipette repeatedly. [BE CAREFUL]. Transfer to a small glass cuvette and add 4 ml of tap water. Uniformly resuspend the blue-stained DNA in the 5 ml solution by pipetting up and down repeatedly. [BE CAREFUL] Your stained DNA solution is highly stable and should be saved for the remainder of the laboratory period. As part of the standard curve analysis, read and record the absorbance of the 5 ml DNA-containing solution at 550 nm.

  17. Assignment for Week 1-2 • Complete and turn in the form on p. 7. • Do Library Assignment 1 tutorial. Libr I Quiz on 9/4. • Read Chapters 1 to 4 (4 & 5 for week 3). Test 1., 9/11 (Ch. 1-4). Read Diversity Lab instructions before coming to class on 9/11. • Finish reading the Scientific Experimentation: Standard Curve Analysis for DNA Quantitation including Writing Lab Report 1 and data analysis (pp. 12-17). • Files on How to Write a Scientific/Lab Report are for your reference. Please follow these useful instructions when you write the three lab reports. • Purchase and turn in one pack of AccuScan #815-E or equivalent by 9/4. • Have textbook for study. Start the Threaded Discussion participation.

  18. Thank you for keeping the lab clean, posting TD and your Facebook • Test #1. 6:45 – 7:25. Back @ 7:30. • Turn in signed p. 7 of syllabus. • Turn in one pack of scantron. Put your name on the Post-it. • Go over lab today. 2-3 per group. • Set up standard curve, measure your DNA sample. • Enter your group’s standard curve date in Excel in front. • See Announcement about Week 2 Assignment.

  19. Standard Curve PreparationApplications • Protein/DNA determination • Enzyme assay • Immunoassay/ELISA • Radioimmunoassay • Virtually all clinical assays

  20. A Standard Curve Grades SD: Standard Deviation 5 10 15 20 Times Wasted in Studying Bio 21 Per Week (hrs) n = 352

  21. 2 beakers 2 pipet bulbs 5 test tubes Test tube rack 1 wax pencil Materials for tonight’s lab Kimwipes 5 Cuvettes Spectrophotometer 1 ml, 5 ml, 10 ml pipets

  22. Materials for tonight’s lab 2 beakers 1 pipet bulbs 3 pipets 5 test tubes Test tube rack 1 wax pencil Spectrophotometer

  23. Intro to Spectrophotometerp. 2 • Beer’s Law: the concentration of a substance in solution is directly proportional (linear, p. 2) to the amount of light absorbed by the solution, and inversely proportional to the log of the fraction of light transmitted by the solution. • The incident light is monochromatic (light of a single wavelength. P. 23 wavelength vs. color

  24. UV spec Radiation spectroscopy Light dispersed by a grating into a continuous spectrum of colors. The wavelength is adjusted by rotating the grating so that a small selected region of the visible spectrum passes through the exit slit into the sample. Light of one wavelength (and color) is passed through a sample of solution and than illuminates a phototube detector. The detector produces an electrical signal corresponding to the light intensity. The magnitude of the signal is displayed on a meter.

  25. DNA Quantitation Absorbance @ 550 nm 0 .002 .004 .006 .008 .001 Methylene Blue Concentration (%) Figure 1. Standard Curve Showing The Relationship Between The Concentration Of Methylene Blue And Absorbance At 550 nm. Ten different dilutions of Methylene Blue were prepared by ??? Each solution was read ??? The curve will be used for ???

  26. Methylene Blue goes into waste chemical bucket.When you are done, • Enter your group’s data when done, they will be posted tomorrow. • Clean up everything that you have used on your bench • Wash and invert all test tubes • Do not forget to take the cuvette out of the spec

  27. Wavelength, use 550 nm Sample well For 0% T, empty For 100% T, use water

  28. Calibration Cuvette with water: 100% transmission (0% absorption) Nothing in sample slot: 0% transmission (100% absorption)

  29. Be absolutely certain about calibration, reading absorbance, using significant numbers, accuracy, and precision before doing experiment

  30. Reading Reading is 0.239 INCORRECT If reading is >1, the reading can only be e.g., 1.13 not 1.134, why?

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