CHEM L333

Chemistry L333: Advanced Biochemical Techniques Spring 2008 Syllabus'Bold text
Archer 631, Mondays 1-5pm and Fridays 1-5pm

Professor: Dr. Melanie Berkmen

Email: mberkmen@suffolk.edu

Phone: 617-973-5321

Office: Donahue 513 (but often found in lab Archer 631)

Office Hours: Tues. 12-1(D513), Thurs 3-4pm (A631), Friday 11-12 (A631) or by appointment. You are always welcome.

Reading (prior to class) will be assigned from:

1. “FT”: Farrell & Taylor, Experiments in Biochemistry: A Hands On Approach, 2nd ed., 2006, Thomson Brooks/Cole. 2. “SB”: Stryer, Berg, & Tymoczko, Biochemistry, 6th ed., 2007, WH Freeman & Co.. 3. Handouts. 4. Journal articles.

Assignments:

Pre-lab quizzes (given at the beginning of class) Lab notebook entries (required for all actual labs but not discussions/journal club) Post-lab problem sets ~5 Lab reports (required for labs 5/6, 7, 8/9, 10-14, 10-18) Final paper on independent research project

Tentative Grading: Lab reports 25% Post lab questions/problems 20% Pre-lab quizzes 20% Final paper on independent research project 15% Laboratory technique, safety, participation, and courtesy 10% Lab notebook 10%

Absences/Missed Assignments: Absences due to a health, family, or personal emergency can be made up. In addition, if you know you will be absent due to a professional activity (such as attendance of a scientific conference or interview), please notify the professor as early as possible to schedule a make-up. All other absences cannot be made up, and will result in no-credit for the missed laboratory and the accompanying quizzes and assignments. Late assignments will lose 4 percentage points for each day that they are late.

Please come to lab on time and prepared.

Three GOALS: A. Acquire skills in basic modern biochemical techniques (1st half of semester): - Pipeting and preparation/sterilization of buffers - DNA/protein purification and electrophoresis - PCR and other biochemical assays

B. Understand the basic process of scientific discovery in the laboratory by development of an independent experimental research project (2nd half of semester) with special focus on experimental design and troubleshooting.

C. You will start as my protégés and end as my colleagues.

TENTATIVE CALENDAR: Part I Basic Biochemical Techniques

Date (Lab#) Pre-lab reading Lecture Topics Laboratory Exercise Post–lab assignment (Due 1 week after lab) 1/18 FRI Introduction, lab tour and safety Notebooks & lab reports How to search/cite literature Biochemistry review & concept mapping Math assessment (not for grade) 1/21 MON NO CLASS – Martin Luther King Holiday 1/25 FRI Lab 1 FT p1-28 Data and measurements Pipette tip packing and sterilization Calibration and use of pipettes Problem Set 1/28 MON Lab 2 FT p37-51 Acids, Bases, and Buffers Calculating dilutions and solutions Purified Water and sterilization Making buffers Problem Set 2/1 FRI Lab 3 SB p134-144 Handout Cloning and plasmids Purification of Plasmid DNA Preparation of an agarose gel 2/4 MON Lab 4 FT p239-242 Agarose Gel Electrophoresis Problem Set 2/8 FRI Lab 5 Handout SB p65-77 Enzyme purification & Pfu DNA Pol Ni2+ affinity purif. Of His- Pfu DNA Pol 2/11 MON Lab 6 FT p239-250 Weber & Osborn, JBC, 1969 Polyacrylamide gel electrophoresis Discussion of Weber & Osborn, JBC, 1969 Graphing with Excel SDS-PAGE of Pfu prep/Coomassie Lab report (on labs 5/6) DUE 2/18/08 2/15 FRI Lab 7 FT p63-73 Spectrophotometry & Beer’s Law Bradford Assay & Calibration curves Bradford Assay Lab report (on lab 7) DUE 2/25/08 2/18 MON NO CLASS – President’s Day Holiday 2/22 FRI Lab 8 FT p89, p94-95, p359-366 PCR, experimental design and controls PCR to assay activity of purif. Pfu DNAP 2/25 MON Lab 9 Agarose gel of PCR products Lab report (on labs 8/9) DUE 3/3/08

TENTATIVE CALENDAR OF LABS: Part II Project Lab

Date (Lab#) Pre-lab reading Lecture Topics Laboratory Exercise Post–lab assignment (Due 1 week after lab) 2/29 FRI Lab 10 HANDOUT Journal Paper Explanation of project and project lab Discussion of Berkmen et al, in prep. Choose mutations 3/3 MON Lab 11 HANDOUT Journal paper Principles of primer design Design and order primers 3/7 FRI Lab 12 FT p313-330 Prep plasmid vector 3/10 MON Lab 13 HANDOUT PCR mutations 3/14 FRI Lab 14 HANDOUT QIAQUICK PCR products Run PCRs and plasmid on agarose gel Guestimate DNA concentrations from gel Lab report (on labs 10-14) DUE 3/24/08 3/17 MON NO CLASS – SPRING BREAK (Troubleshoot PCRs?) 3/21 FRI NO CLASS – SPRING BREAK (Troubleshoot PCRs?) 3/24 MON Lab 15 FT p295-302 p316-322 HANDOUT Restriction digests of PCR and plasmids Qiaquick digests 3/28 FRI Lab 16 HANDOUT Run digests on gel Gel purify vector 3/31 MON Lab 17 HANDOUT Discussion of ligation and transformation Controls for both Ligate vectors and PCRs Transform 4/4 FRI Lab 18 HANDOUT Analysis of Transformations Colony PCR to check for inserts Lab report (labs 10-18) DUE 4/7/08 4/7 MON Lab 19 HANDOUT Run insert-check PCRs on gel 4/11 FRI Lab 20 HANDOUT Plasmid prep – elute with water Send for sequencing 4/14 MON Lab 21 HANDOUT BT p138 Analysis of sequences Comparison of sequences: Clustal, Blast Calculation of mutation rates 4/18 FRI Lab 22 HANDOUT Bioinformatics – viewing protein structures, navigating NCBI, etc. In class problem set In-class concept mapping Problem Set FINAL Paper (on labs 10-22) DUE 4/21/08 4/21 MON NO CLASS – Patriot’s Day