LABORATORY SCHEDULE - Fall 2009

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8:30

Welcome / Orientation:  CGRC room 184

8:30

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8:45

Lab: Course overview and objectives (Dr. Bill Farmerie and Sharon Norton)

8:45

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9:30

Lecture: Carbonic Anhydrase Model (Dr. David Silverman).

8:45

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10:15

Lecture:  Introduction to Protein Chemistry (Dr. Sixue Chen).

10:15

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11:30

Exp. 2: Lysis of E.coli .
Exp. 4: Affinity purification up to sample incubation.

11:30

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12:15

Lunch (on your own).

12:15

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1:15

Lab: Bioinformatics I: Introduction to Web-based Tools (Dr. Fahong Yu)

1:15

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2:15

Exp. 3 A,B,C: SDS-PAGE analysis of bacterial lysate fractions

2:15

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3:45

Exp. 4 cont., Exp. 5 and Exp. 11: Affinity purification, de-salting and concentration by ultrafiltration, 2D gel sample prep up to -20° incubation.

3:45

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5:00

Exp. 3D: Gel Staining and De-staining overnight

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9:15

Exp 6:  Protein concentration assay.

9:15

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9:30

Exp. 3E: Gel Drying

9:30

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10:15

Exp. 8: Minigel apparatus (Sharon Norton). Pour sep gel.  Tris-glycine system.

10:15

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11:00

Lecture: Theory of Electrophoresis.  ( Dr. David Moraga).

11:00

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11:30

Exp. 8: Pour stacking gel

11:30

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12:15

Lab: Bioinformatics II: Excel for Protein Concentration and Enzymatic Activity (Dr. Fahong Yu)

12:15

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1:00

Lunch (on your own).

1:00

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1:45

Exp. 8: Prepare, load samples, electrophorese.

1:45

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3:00

Exp. 9A, B, C:  Protein transfer. Block colormetric blots O/N, put chemiluminescent blots in wash buffer 1, ON.

3:00

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3:30

Exp. 11A: Finish sample preparation and set up O/N rehydration of two-dimensional gel electrophoresis sample.

4:00

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5:00

Graduate Students Reception: Founders Gallery

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9:00

Exp. 9C:  Add 1st Ab to immunoblots, both chemiluminescent and colormetric.
Exp. 11B: 1st dimension of 2D electrophoresis (Includes demo of instrument set-up).

9:00

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10:00

Lecture: Winning Westerns (Linda Green).

10:00

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11:00

Exp. 9C: Wash Nitrocellulose memebrane/Add 2nd Ab for colormetric, finish chemiluminescent.

11:00

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12:00

Lecture: 2D Gel Electrophoresis (Dr. Sixue Chen)

12:00

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1:00

Lunch

1:00

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2:00

Exp. 11C,D: Equilibration of IEF strips and begin second dimension.

2:00

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3:00

Exp. 9C Finish Western blots (wash, color substrate addition)

3:00

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4:30

Exp. 11D,E: Disassemble 2nd dim. gel and stain. De-stain in water, ON.

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9:30

Seminar: Animal Awareness ( Charles Mason)

9:30

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10:30

Exp. 12A: Preparation of sample through washing of gel pieces.

10:30

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11:15

Lecture:  Biological Mass Spec Overview and Sample Preparation (Dr. Sixue Chen).

11:15

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12:30

Exp. 12A : Reduction and Alkylation of Proteins

12:30

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1:30

Lunch

1:30

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2:30

Lecture: Bioinformatics III, Introduction to Bioinformatics tools (Dr. Fahong Yu).

2:30

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3:30

Exp. 12A: Trypsin digestion and extraction (microwave method).Note: This will be done by Lab Staff during the lecture.

3:30

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4:30

Exp. 12B and C: Demo of preparation of matrix and zip tips.
Exp. 12D: Demo of mass spec and Proteomics Lab tour.

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11:30

Exp. 14A: RNA Isolation and spectrophotometric analysis.

11:30

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12:15

Exp. 14B: Cleaning of apparatus and pouring of gel. Preparing the samples and loading the gel.

12:15

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1:00

Lunch

1:00

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2:00

Lecture and Demo: RNA Isolation and QC: Bioanalyzer (Gigi Ostrov, Ph.D. and Yanping Zhang Ph.D.)
Exp. 14: Photograph RNA gel

2:00

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3:00

Lecture: Bioinformatics IV-Designing your own primers for the RT-PCR of mouse CAII (Dr. Fahong Yu).
Lab: Designing your own primers and optimization experiment for the RT-PCR of mouse CAII

3:00

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4:00

Exp. 15: Set up reverse transcription and PCR reaction.

4:00

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4:30

Exp. 12: Mass Spectrometry analysis: Database searching demo and computer time (guided by Carolyn Diaz).

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9:00

Exp.16: Gel electrophoresis of RT-PCR samples.

9:00

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10:00

Lecture: PCR Theory (Dr. Bill Farmerie).

10:00

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12:00

Exp. 16A,B,C: Excise bands. Purify DNA from gel slice. Pour gel.

12:00

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1:00

Lunch

1:00

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2:00

Exp. 16D: Start TOPO ligation system (bacterial transformation).
Exp. 18 A: NEBlot Phototope probe labeling, up to 37° incubation.

2:00

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3:00

Lecture: Gene Cloning (Dr. David Moraga)

3:00

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4:00

Exp 16D: Spread X-gal on agar plates. Plate cells.
Exp. 18B: MinElute probe purification.

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12:00

Exp. 17A: Begin gDNA isolation (up to 100% ethanol precipitation).

12:00

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12:30

Lecture: Hybridization Techniques (Sharon Norton)

12:30

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1:45

Lunch

1:45

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2:45

Exp. 17A: Finish DNA isolation, pour gel.

2:45

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3:15

Exp 17B: Agarose gel analysis of genomic DNA

3:15

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4:15

Lecture: Bioinformatics V-In Silico cloning and screening (Dr. Fahong Yu).

4:15

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4:45

Exp 17B, C: Quantitate genomic DNA, set-up incremental digestions

4:45

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5:00

Exp. 16E: Begin overnight cultures from individual bacterial colonies.

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9:00

Exp. 17D: Agarose electrophoresis of gDNA for Southerns.

9:00

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10:00

Exp. 16E2-E3, F: Plasmid miniprep, set up restriction enzyme digestions.

10:00

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10:30

Exp. 20: set up cDNA for real time PCR

10:30

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11:15

Lecture: Real-Time PCR (Sharon Norton)

11:15

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12:00

Exp. 20: Set up real-time PCR

12:00

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12:45

Lunch

12:45

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1:00

Exp. 17D: Lab assistant will take photograph of digested DNA gels.
Exp. 16G: Gel electrophoresis.

1:00

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3:00

Exp. 17E: Set up Southern Transfer.
Exp 16G: Photograph gel.

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11:30

Exp. 17E-17F1: Dismantle Southern, UV-crosslink, begin prehyb.

11:30

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12:00

Lab : Real time PCR data review

12:00

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12:30

Exp. 17F1: Denature biotin-labeled probe and begin hybridization.

12:30

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1:30

Lunch

2:00

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3:00

Lecture: Practical Aspects of DNA Sequencing with 454 and Solid (Dr. Ernie Almira)

3:00

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5:00

Exp. 17F1, F2: Finish Southern hybridization.

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5:00

Bioinformatics Project Time.

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5:00

Bioinformatics presentations: Lunch Provided