To open protocols as a printer-friendly format, go to http://lowepowerlab.ucdavis.edu/protocols/

Protocols

Lab protocols for the Lowe-Power lab

Lab Management & Maintenence

Important information for all full-timers!

• Chemical Management
• Purchasing
• What to do if the building loses electricity

Undergrad Relevant Information

• Preparing sterile supplies
• Shelf stocking
• Autoclave

Familiarize Yourself with Common Lab Terms

Lowe-Power Lab Glossary

This link gives access for viewing and leaving comments if you have questions or want to suggest changes.

Molecular Biology

• DNA Electrophoresis
• Plasmid and DNA sequence management in the lab Benchling folder
• PCRs to detect & classify Ralstonia
• Primer Design with PrimerBlast
• Overview of cloning tools for Ralstonia
• Gene deletion in *Ralstonia *
• Gibson assembly
• Colony PCR

• Step-by-step guide to gene deletion (protocols.io by Noah Guillome):

  • Unmarked Gene Deletion Ralstonia Primer Design. If you are new to genetically engineering in our lab, please have Tiffany approve your primer choices. This will be an opportunity for her to teach you more about bacterial genetics (or learn more about bacterial genetics from you)
  • Upstream&Downstream PCR Amplification for Unmarked Gene Deletion Ralstonia
  • Gibson Assembly for Unmarked Gene Deletion Ralstonia
  • Colony PCR for Unmarked Gene Deletion Ralstonia
  • Ralstonia Electroporation for Unmarked Gene Deletion
  • Using PCR with external primers to confirm knockout mutants, for Unmarked Gene Deletion Ralstonia
• Sanger Sequencing (We tend not to do Sanger sequencing anymore, and go with whole-plasmid sequencing with Plasmidsaurus)

In addition to our lab protocols, here are resources that are very useful:

• A Primer on Cloning in E. coli by Michael Nonet at Wash U

Microbiology

• General growth of Ralstonia
• Glycerol stocks (-70C)
• Media recipes
  • [] Add table of contents
• [draft] Buffers and Reagents
• Antibiotics
• Chemically competent E. coli - Mix&Go
• Electrocompetent Ralstonia cells
• Natural transformation of Ralstonia
• Conjugation with E. coli WM3064
• [] Growth curve: test tube
• Growth curve: 96 well microplate
• Dilution plating & colony forming units
• Competition Assay This is written for an in planta assay, but could be adapted to in vitro experiments.

Plants

• Tomato: sowing, growing, & transplanting
• Tomato: petiole inoculation
• Tomato: soil soak inoculation
• Tomato: xylem sap harvesting by root pressure
• [] Melon: inoculations
• Measuring wilt symptoms for a disease progress assay
• Colonization: tomato stem
• Competition Assay This is written for cut-petiole competitions, but it can be adapted to soil soak.
• [] Colonization: tomato roots
• [w.i.p.] Growing Tomato for seed
• [] Tobacco Mosaic Virus & other things to worry about

Workbooks and Excel templates

• (Gibson) assembly
• bacterial density
• chemistry calculations
• PCR
• 96- and 24-well microplate templates

KBase and Bioinformatics

• Useful KBase narratives

GitHub

• Best Practices for Markdown / editing protocols
• [draft] Git and Github e.g. How to Update lab protocols with forking, committing, and pushing

References & Acknowledgement (and great resources)

This repo is inspired by Mark Mandel’s lab protocol repo. What a gem. Additional labs with great protocol repositories are Barrick lab, ..

I also highly recommend using the AddGene website as a resource on molecular biology. http://www.addgene.org/mol-bio-reference/

This work is licensed under a Creative Commons Attribution 4.0 International License.