WARNING NOTICE
The experiments described in these materials are potentially hazardous and require a high level of safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented.
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Research Projects
You will be doing real research in this lab. Each research project will center on examining the biology of Pseudomonas aeruginosa. However, each team will be designing their own laboratory experiments to research different aspects of the bacterium. Students will choose from a list of possible projects (PDF).
Lab Notebooks
Your notebook should be a complete record of all your experiments as they were actually performed. A good notebook will enable someone to reconstruct, long after the fact, exactly what was done and why.
Information on the Experimental Microbial Genetics Lab Notebook (PDF)
Protocols
The following is an index of laboratory protocols that were used by students in this course.
| PROTOCOL NAMES | DOCUMENTATION |
|---|---|
| Agarose gel electrophoresis and DNA band excision | (PDF) |
| Antarctic phosphatase | New England biolabs antarctic phosphatase |
| Biofilm plate assay | (PDF) |
| Care and handling of PA01 fur mutants | (PDF) |
| CAS agar plates for pseudomonas — high and low fe | (PDF) |
| Current protocols online | Wiley interscience protocols |
| Characterization of phenazines in pseudomonas | (PDF) |
| CIP — calf intestinal alkaline phosphotase | New England bioLabs alkaline phosphatase |
| Datsenko 2000 PNAS red swap | Datsenko, K. A., and B. L. Wanner. "One-Step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products." PNAS 97, no. 12 (2000): 6640-5. ( PDF) |
| Dephosphorylating with CIP & T4 ligation reactions | (PDF) |
| Designing primers for PCR | (PDF) |
| DIC & fluorescence microscopy | (PDF) |
| Generating and screening for mutants in fur | (PDF) |
| IQ SyBR green supermix | (PDF) |
| Lysozyme | (PDF) |
| MegaX DH10B T1R electrocomp cells | (PDF) |
| Motility assays | (PDF) |
| NEB quick ligation kit | New England biolabs quick ligation kit |
| NEB T4 DNA ligase | New England biolabs T4 DNA ligase |
| PA14 Tn mutant library | (PDF) |
| PCR with platinum PCR super mix | (PDF) |
| Plate cell counts | (PDF) |
| Pseudomonas electroporations | (PDF) |
| Qiagen hispeed plasmid midi and maxi kits | (PDF) |
| Qiagen miniprep kit protocol | (PDF) |
| Qiagen QIAquick gel extraction protocol | (PDF) |
| Qiagen QIAquick PCR reaction & enzyme purification kit protocol | (PDF) |
| Restriction enzyme digests | (PDF) |
| Ribonuclease A | (PDF) |
| SAP — shrimp alkaline phosphotase | (PDF) |
| Spectrophotometric determination of DNA concentration | (PDF) |
| T4 polynucleotide kinase | New England bioLabs T4 polynucleotide kinase |
| Tips on how to troubleshoot your cloning | (PDF) |
| TOPO TA PCR cloning kit | (PDF) |
| Transformation of DH5a-T1R chemically competent cells | (PDF) |
| Epicentre failsafe PCR kit |
Epicentre biotechnologies failsafe PCR premix selection kit (PDF)
|
Archives
You will be required to archive the strain and plasmids you construct during the semester.
- Guidelines for archiving with strain and plasmid list template (PDF)
- Archive example for plasmid sequence and map (PDF)