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) |
| 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) |
| 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) | |
| Lab 4 | Prepare protein purification buffers. Create a BSA standard curve for future protein quantification | Lab Session 4 (PDF) | |
| Lab 5 | Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS | Lab Session 5 (PDF) | |
| Lab 6 | Prepare an SDS-PAGE protein gel | Lab Session 6 (PDF) | |
| Lab7 | Run SDS protein gel. Concentrate protein and quantify final protein concentration | Lab Session 7 (PDF) | |
| Lab 8 | Lab Session 8 (PDF) | ||
| Lab 9 | Set up PCR for DNA mutagenesis | Lab Session 9 (PDF) | |
| 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) | |
| 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) | |
| Lab 15 | Complete crystal structure viewing exercises. | Analyze DNA sequencing results |
Lab Session 15 (PDF) Key (PDF) |
