5.36 | Spring 2009 | Undergraduate

Biochemistry Laboratory

Labs

Complete student manual (PDF)

Supplemental TA instructions (PDF)

Table of contents (PDF)

Appendix A-D and references (PDF)

Equipment and supplies list (PDF)

The table below contains links to the instructions for each lab session, as well as links to photographs of the labs.

SES # H396P ABL PROTEIN EXPRESSION/ KINASE INHIBITION ASSAYS DNA SITE-DIRECtED MUTAGENESIS LAB MANUAL
Lab 1

Grow a starter culture of cells with the H396P Abl and Yop-encoding vectors

Grow a starter culture of cells with the wild type Abl vector

Lab Session 1 (PDF)

Photos

Lab 2

Express the H396P Abl protein. (Spin down cells on the following day.)

Isolate wt-Abl vector DNA through a miniprep. Quantify DNA concentration by UV-Vis

Lab Session 2 (PDF)

Photos

Lab 3   Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant Lab Session 3 (PDF)

Photos

Lab 4 Prepare protein purification buffers. Create a BSA standard curve for future protein quantification   Lab Session 4 (PDF)

Photos

Lab 5 Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS   Lab Session 5 (PDF)

Photos

Lab 6 Prepare an SDS-PAGE protein gel   Lab Session 6 (PDF)

Photos

Lab7 Run SDS protein gel. Concentrate protein and quantify final protein concentration   Lab Session 7 (PDF)

Photos

Lab 8 Lab Session 8 (PDF)

Photos

Lab 9   Set up PCR for DNA mutagenesis Lab Session 9 (PDF)

Photos

Lab 10   Complete the DPN digest and transform storage cells with mutant DNA. Pour LB/agar plates Lab Session 10 (PDF)
Lab 11   Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing Lab Session 11 (PDF)

Photos

Lab 12 Prepare buffers and reagents for the coupled kinase activity assay   Lab Session 12 (PDF)
Lab 13-14 Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors   Lab Session 13-14 (PDF)

Photos

Lab 15 Complete crystal structure viewing exercises. Analyze DNA sequencing results

Lab Session 15 (PDF)

Key (PDF)

Course Info

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Spring 2009
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