This section contains documents that are inaccessible to screen reader software. A "#" symbol is used to denote such documents.
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.
Lab files.
| 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 |
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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)
|