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Biochemistry 452

Biochemistry 452

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Biochemistry 452

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  1. Dennis T. Brown Room 346 Polk Hall Phone 515 5765 E-mail: dennis_brown@ncsu.edu Biochemistry 452

  2. E-mail • Major mechanism of communication • Notify you of changes in schedule and project • Send updates on experiment results • Answer your questions • Keep your mail box clean • Lecture notes on web as too large to send • Some Mails may still push you over quota

  3. Lab Text • BCH 452 “Experimental Biochemistry, by Hardin-Ascencio-Knopp, Kendall-Hunt Eds. ISBN 978-1-4652-1311-2. Available at the University Bookstore or at Amazon.com (http://www.amazon.com/Experimental-Biochemistry-HARDIN-CHARLES/dp/1465213112/ref=sr_1_1?ie=UTF8&qid=1386083987&sr=8-1&keywords=experimental+biochemistry%2C+hardin)

  4. Lecture Notes and other info posted on Biochemistry web • http://biochem.ncsu.edu/faculty/brown/brown.php • At top near photo click on “Biochemistry 452 Notes” • Look at Powerpoints prior to lab

  5. Schedule • Lab opens at 1:15 • Turn in Pre lab questions & lab reports • Collect any handouts • 1.30 lecture • 2:00 lab exercise • Finish usually around 5:00

  6. NO MAKE UP LABS • Late assignments without a valid excuse will not be accepted and will receive a score of zero. • Incomplete as a course grade will be awarded only for work not completed during the course due to conditions deemed by the instructor to be beyond the reasonable control of the student. • Attendance to all laboratories is mandatory and unexcused absences will affect the outcome of the course. A reduction of 10 points per missed laboratory session will be applied to the final grade for unexcused absences.

  7. Bch 452 • Will introduce fundamental techniques in biochemical analysis • Will teach the principles upon which the technology depends • Will make you familiar with theory and practice of the technology

  8. More Importantly BCH 452 • Will stress proper laboratory technique • Reproducibility • Eliminate errors • Will stress proper record keeping and data reporting • Necessary for archival considerations • For intellectual property • For trouble shooting

  9. Concept of 452 • Teach techniques in common use in biochemistry laboratory science • Try to present situations that exist in a real laboratory situation • Move from “exercises” with predictable outcomes. • To “experiments” with doubtful outcomes • Important to understand and explain anomalous results

  10. My Job • To be certain that you understand the principles that the experiments will examine • To be certain that you understand how the apparatus employed in the experiment functions • To be certain that you understand the reason for each step in the experimental protocol

  11. Lab Safety (Page XXIII) • Students are expected to observe proper laboratory procedures as described in the lab manual and should sign the disclosure on the first day of lab (Page XXVII) • a. This is a serious aspect of lab science. Go through the list in the manual. • b. Read each laboratory experiment before you come to class. • c. Follow precautionary statements given in each experiment. • d. Only necessary work materials should be at or on the laboratory bench. Do no place coats, backpacks, or other personal belongings on your lab bench; store them in the lab cabinets or on the coat rack. Do not place them in the floor. • e. Do not eat, drink, smoke or place materials in your mouth while in the laboratory. • f. Wear appropriate clothing and eye protection while in the laboratory and experiments are in progress NO OPEN TOE SHOES • g. Report all accidents to the instructor or TA immediately: such as spills, cuts, burns, or contact between reagents and your body or eyes. Take appropriate steps in an emergency situation (such as the use of the eye wash station or emergency shower).

  12. Hazardous materials • Hazardous reagents • Volatiles • Corrosives • Mutagens • Material Safety Data Sheets (MSDS) • Important information regarding all chemicals • Example on page XXV

  13. Model Molecules • The three major classes of biomolecules • Carbohydrates • Proteins • Nucleic acids

  14. Model Techniques • Spectrophotometry • Centifugation • Chromatography • Electrophoresis • Biochemical fractionation • PCR

  15. Your Responsibility • Read the experiment before you come to lab • Read pertaining sections of appendix • Read “Info Boxes” • Turn in the pre lab exercise • Have all required materials • Some of the protocols can be confusing. Make a flow sheet that outlines the steps.

  16. Grading • Lab reports: 130 total points • Pre Lab Questions: 33 points • Exams (2): 25 points each plus 3 free points • Total 216 points • A+ 216 - 205 C+ 150 - 140 • A 204 - 194 C 139 - 130 • A- 193 - 184 C- 129 - 119 • B+ 183 - 173 D 118 - 108 • B 172 - 162 F 107 - 0 • B- 161 - 151

  17. Extra Credit • 1. An extra 10 points is added to the Protein Purification and Southern blot Lab reports due to increased write-up requirements. • 2. Lab C1: 5 points extra credit for Arrhenius plot and activation energy calculation (see page 94) • 3. 7 extra credit points possible for a well-kept lab note book

  18. Reporting • Prelab questions • Clearly indicated (see page 28 for next weeks lab) • Due at beginning of designated lab • Ensures that you have read and understand the exercise • Completed report • Due at beginning of next exercise • Report for Lab A1 due at start of lab C1

  19. Laboratory Notebook (ideal) See Page 3 • No loose pages • Generates copies • All entries dated • Each page dated • Pages signed by a witness attesting to date (extreme) Why?? • Retention time (7 years) Why ??

  20. Lab Reports (Page 4) • Should contain • Statement of purpose • Why you are doing experiment • What you wish to determine • Brief account of theory and design of experiment • Summary or flow chart • The raw data • All calculations • Results • Conclusions and interpretation

  21. A form for lab write up in lab manual • Title all sections • Abstract, Discussion, Graph Titles etc. • Abstract should contain a concise summary of experiment • “I investigated the effect of temperature on activity of the enzyme Stickase by monitoring its ability to convert stick to broken stick. This was done by determining the accumulation of broken stick by spectrophotometry (A260) at temperatures between 10 to 60 degrees C. I found that Stickase converted stick to broken stick most efficiently at a temperature of 37oC”

  22. Lab Report • Record all your experimental data including changes in protocol • Label co ordinates. Be careful with units, spelling and grammar • Explain sources of error • It is not as important that you get a “correct” outcome as it is that you can explain your result.

  23. Remember • Your lab report is your PERSONAL representation of experimental results • Because you work in groups the raw data produced will be identical however your report should be distinct.

  24. Things that you should know

  25. Units of Biochemistry • Decade system • Moles • Meters • Liters • Grams • Centigrade

  26. Elementary calculations • Gram Molecular Weight • Dilutions • Rate equations • “What is the molar concentration of a solution that is 5 millimolar NaCl”

  27. Significant Figures • Determines the value of a data set • Includes estimations • 3.2 is not the same as 3.20 or 3.200

  28. Presentation of Data • Data tables • Graphs

  29. A table or Graph Should: • Be easy to understand • Project a message • Does not require extensive reference to text

  30. Experiment • A solution of compound X is reacted with reagent Y which causes the production of a light absorbing product. The amount of light absorbing product indicates how much compound X is present. The sample containing X is divided into three aliquots and the experiment is repeated three times at three wavelengths.

  31. Lousy Table

  32. Good Table

  33. Bar Graph A: Compares Values at each wavelength, Shows Reproducible

  34. Bar Graph B: Compares absorbance at different wavelength, shows sensitivity

  35. Next Exercise • Carbohydrates and standard curve • Section A1 in lab manual (page 26) • Read carefully prior to coming to lab. • Do the pre lab questions turn them in on the 13-14th

  36. Benchtop Protocols • Page 387 • The benchtop protocol summarizes step be step the days experiment. It is more convenient than having the entire manual open on your bench

  37. DVD • The DVD shows step by step each part of the experimental protocol, watching it will help prepare you for the days exercise