CHEM 319 BIOCHEMISTRY LABORATORYFALL 2012Dr. WU XU

Lecture 1: Introduction of Biochemistry Laboratory

Course Description, Grading, Laboratory Report and Notebook

Student background form (due today)Laboratory Safety and Rules (due today)Protein selection for yeast two hybrid experiment (due

today)Assign groups (due today)Check in (form is due today)Pipette: P1000, P200, P100, P20 and P10Introduction of Experiment 1

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Contact Information

Dr. Wu XuOffice: Montgomery Hall Room 138Email: wxx6941@louisiana.eduTelephone: 337-482-5684Office hour: Tuesday & Thursday 10:00 – 11:00 amStudent Background Form

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Course Description and ObjectivesWelcome to Biochemistry Laboratory! This course (Chem319) is designed to introduce students to basic biochemical techniques commonly used in a research laboratory. Students will have the opportunity to acquire the experience working with four major classes of biomolecules: carbohydrates, proteins, lipids and nucleic acids. Techniques include measurement of pH, spectrophotometry, centrifugation, isolation and purification of carbohydrates, lipids, proteins and nucleic acids, chromatography, enzyme kinetics, electrophoresis and polymerase chain reactions. The lab will enable students to correlate theories and concepts studied in lectures to experimentation and data collected in the lab.

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Course Description and Objectives Continued

Students completing this course should be able to:Perform a variety of common biochemical experiments with

adherence to accepted standards of accuracy and safety;Collect reproducible and interpretable data;Become familiar with basic computational and statistic aspects

of data analysis;Explain the meaning of the results;Maintain records in a laboratory notebook and prepare research

reports following professional standards; Become acquainted with the lab’s procedures dealing with

emergency and safety situation as well as procedures for managing chemicals, equipment and waste.

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Course Requirements

Prerequisite or Coprerequisite: Chem 317

Prerequisite or Coprerequisite: Chem 233

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Laboratory Safety and RulesSafety in the laboratory is of utmost importance and there will be no tolerance of safety infractions. Failure to follow safety rules will result in dismissal from the laboratory.

Eating, drinking and smoking are not permitted in the laboratory. Do not put anything in the laboratory into your mouth. Students will never, under any circumstances, pipet by mouth;

Personal belongings are not allowed on the laboratory benches. Please deposit all personal belongings under the assigned working tables in the laboratory;

Use of cellular phones is not permitted. Please turn off cellular phones before entering the laboratory;

Always thoroughly wash your hands before leaving the laboratory;Dress: you will be asked to leave if you are not dressed appropriately:

• Safety glasses or goggles must be worn at all times. • Shoes (i.e. closed toes), not sandals, are required. • Students are strongly advised to wear a laboratory coat and gloves. • Short shorts or short skirts are not allowed unless covered completely.• Ladies: tie long hair behind the head when working with Bunsen burners or reagents so the hair does not fall in front of the face or into liquids.

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Laboratory Safety and Rules ContinuedEquipment: please do not attempt to use any equipment until you have read

and understood the operating instructions, or have been given appropriate instructions. Don’t hesitate to ask the instructor if you have questions;

Label the contents of all tubes, flasks, beakers, etc. It is easy to forget or mix things up;

Wash all glassware and clean your work area prior to leaving; Waste disposal:

• Do not put hazardous waste down the drain. No organic liquids except ethanol should be put down the drain. Water solutions are OK to pour down the drain unless you are told differently. Waste containers will be provided for substances that require special disposal. Check with the instructor if you have any questions about disposal. • Do not put solid or powdered chemicals into ordinary waste cans. There are marked containers for solid wastes. • Do not put anything that is sharp or can be broken into ordinary waste cans. All disposable glass and anything that is sharp should be placed in a specially marked ‘SHARPS BOX’ to prevent harm to the housekeeping staff.• In general, keep compatible wastes together. That is, do not mix liquids with solids, organic liquids with water solutions, or acids with bases and the like.

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Laboratory Safety and Rules ContinuedGeneral Guidelines:

• Familiar yourself with the laboratory’s procedures; • Locate the first aid boxes, showers, eyewash fountains, fire extinguishers and other emergency equipment;• Emergency contact: Joey Pons, Environmental Health and Safety Director, at 2-5357 or at safetyman@louisiana.edu.

In an Emergency:• If there is a fire: wet towels, fire extinguishers, activate fire alarm • Personal injury: call physician at once• Chemicals in the eyes: flush with large amounts of water from eyewash fountain• Swallowing of chemicals: call physician and poison control at once• Thermal burns: call physician at once• Shock: keep the victim quiet, lying down and comfortable and call physician• Fainting: keep the victim quiet, lying down and comfortable and call physician• Inhalation of fumes: remove victim to fresh air and call physician at once• Chemical spills: wash with water• Uncontrollable reaction: unplug the electrical apparatus or turn off the power source

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Laboratory Safety and Rules Continued

Attendance is mandatory. There will be no “make-up” labs and quizzes. Please see the instructor if you must miss a lab. A score of “ZERO” will be assigned for each missed lab or quiz.

Please be on time. Each lab period will begin with an important short lecture or a quiz.

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Laboratory ReportEach experiment requires a written laboratory report, which contains:

TitleAbstractIntroductionMaterials and MethodsResultsDiscussionReferences (not required in this laboratory

course)

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Laboratory Report ContinuedAn important part of this laboratory course is to develop the students’ ability to write a formal scientific report:

Title (2 points): a single sentence that states the experiment;Abstract (2 points): concisely state the problem, results and

significant of the results;Introduction (2 points): one or more paragraph that explain the

purpose of the experiment and provide any relevant background information to help readers to follow the narrative of the report;

Materials and Methods (2 points): describe instrumentation, materials, supplies and procedures used in performing the experiment;

Results (20 points): statements of fact regarding the data collected; appropriate tables or figures could be used;

Discussion (10 points): an interpretation of the results

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Laboratory Report ContinuedReports should follow the following style convention (2 points)

Must be typed and not handwrittenDouble spaced, except for tables, which are to be

single spaced12 point font size, Time New Roman for all text but

title, which is 14 point bold. Section headings are 12 point bold

1’’ margin top, bottom, left and rightStudent and partner’s name provide below title,

along with the date experiment was performed

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Laboratory Report Continued

Laboratory reports are due first thing following Wednesday afternoon (the beginning the next laboratory);

Each laboratory reports are worth 40 points;Late reports will not be accepted and will be graded

zero;Although the labs themselves may be performed in

groups, the reports must be individual efforts and are not copied from another student;

If duplicate reports are identified, both parties will receive zero for that lab.

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Laboratory NotebookYou need to purchase a notebook for keeping lab notes. The notebook is different from the written lab report. The purpose of the notebook is allow someone to see exactly how the experiment was performed and what results were obtained. Here are guidelines:

The first two pages must be reserved for a table contents. The table of contents is filled in as the experiments are performed with the date;

Notes must be taken in ink. No pencil;Pages must be numbered sequentially, and under no

circumstances are pages to be ripped out or removed;For each experiment, the student must do the following:

Starting a new page, provide the title and date;State the purpose of the experiment;Show the calculations;Show the procedures, data collected or observations

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Laboratory Notebook ContinuedThe notebooks will be turned in and graded at the end of the course. The notebooks will be graded out of maximum of 40 points.

Table of content is accurate (5 points) (-1 point for each error or missing experiment)Page are sequentially numbered with no missing pages (5 points) (-1 point for each missing page)Each experiment has the date and statement of purpose (5 points) (-1 point for each missing date or statement of purpose)All data is written in ink and firmly attached, no loose scraps of paper (20 points) (-2 points for each lab written in pencil, or each loose piece of paper)Handwriting does not need to be pretty, but a reader should be able to read it (5 points) (-2 points for any lab where the protocol is impossible to decipher

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Quizzes and Final ExamThere will be total 7 quizzes. Each is worth 25

points. The quiz will start promptly at the beginning of the

class and last no more than 25 minutes. There are no make-up quizzes for any reason, this

includes being late for lab.Each quiz will cover the theory and calculations

relating to the experiment performed.Final exam will be worth 100 points. It covers all

experiments performed. A study guide will be given for the preparation for the final exam.

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Academic Dishonesty

We strongly encourage students to work in a small groups to discuss experiment protocols, to gather data, and to discuss the implications of the results. In a few instances, the data obtained will be shared with others in the class.

However, quizzes and lab reports submitted for grading must be an original efforts of the student.

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GradingThe final grade for this course (Chem 319 Fall 2012) will be based on the followings:

Written lab reports: 40 x 7 = 280 points possibleQuizzes: 25 x 7 = 175 points possibleFinal exam: 100 points possibleLab notebook: 40 points possible

The grading is expected to follow a percentage scale:

F<60%D>60%C>70%B>80%A>90%

Grade%

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ExperimentsBasic laboratory skills: pH measurement and bufferCarbohydrates: Isolation lactose from dry milk; Analysis of

polysaccharide composition by thin layer chromatographyProteins: lysozyme isolation from egg white and specific activity

determination; protein determination; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); an enzyme kinetics study; study protein and protein interaction by yeast two hybrid experiment

DNA: plasmid isolation, restriction enzyme digestion and electrophoresis; genomic DNA isolation; polymerase chain reaction (PCR)

Others: quantitative determination of vitamin CUse of internet and supercomputing for biomedical

research and data analysis

Tentative Course Schedule (Chem 319 Fall 2012)

Date Experiment Quiz Report due08/22/2012 Check in, safety, basic laboratory skills No No

08/29/2012 pH measurement No No

09/05/2012 Protein purification Yes Yes

09/12/2012 Protein determination No No

09/19/2012 SDS PAGE No No

09/26/2012 An enzyme kinetics Yes Yes

10/03/2012 Isolation of lactose from milk Yes Yes

10/10/2012 Analysis of polysaccharide composition No No

10/17/2012 Isolation of plasmid and enzyme digestion Yes Yes

10/24/2012 DNA analysis by electrophoresis and PCR No No

10/31/2012 Use of internet and supercomputing in medical research Yes Yes

Tentative Course Schedule (Chem 319 Fall 2012)

Date Experiment Quiz Report due

11/07/2012 Quantitative determination of vitamin C Mammalian cell culture No No

11/14/2012 Mammalian cell culture, cell transfectionand luciferase assay and RNA isolation Yes Yes

11/21/2012 Real-time PCR and RNA analysis Yes No

11/28/2012 One hour final exam and check out N/A Yes

Suggestions for Thinking

Learning is a life-long experience. Study hard and enjoy learning.