General Lab Protocols
- Washing Dishes
- Cleaning with Nitric Acid
- Cleaning with Chromerge
- Running the Wang Lab SpeedVac Centrifuge
Media & Plates
- Pouring Plates
- M9 Stock Solutions – version 1 from the lab website
- M9 Media (using version 1 M9 stock solutions)
- M9 Stock Solutions – version 2 used by Zizi, Tyler, & Melanie
- M9 Media (using version 2 M9 stock solutions)
- MSgg Stock Solutions
- MSgg Media
- 10X MC recipe (B. subtilis transformation medium)
- 10x SPIZ (Spizizen’s minimal salts)
- 10X S750
- Zymomonas mobilis Rich Media (ZRMG)/ZRMG Plates
- Zymomonas mobilis Minimal Media (ZMM) 10X Base
- Zymomonas mobilis Minimal Media (ZMM) version 3
- Zymomonas minimal media for Hungate (ZMH) media v1
- Detection/Isolation Enrichment Media (DEM)/DEM Plates for Zymomonas mobilis
- MTC media (for Clostridium thermocellum and Thermoanaerobacterium saccharolyticum)
- SOB/SOC Media
- WM6026 and Mating Plates
- Antibiotic Concentrations for Medias and Plates
- Phosphate Buffered Saline (PBS) 10X Solution
- Making tubes and serum bottles anaerobic
Cell Protocols
- Gram staining protocol
- Conjugation protocol for Z. mobilis using WM6026 donor strain
- E. coli Chemical Competency Cell Preparation
- Transformation of DH5α E. coli
- Colony PCR and colony PCR template preparation for B. subtilis
- B.subtilis transformation using plasmids – two steps protocol (Noah’s)
- Bacillus subtilis biofilms in 12-well plates
DNA Protocols
- qRT-PCR protocol for B. subtilis (Wang lab)
- Modified Genomic DNA Extraction (From Dave)
- TBE electrophoresis buffer preparation (10X)
- PCR transformation protocol for B. subtilis
Molecular Cloning Guide
- The Big Picture: Cloning into Zymomonas
- Two approaches to cloning
- Screening for successful, correct plasmid construction
- Construct Design
- Designing Gibson Cloning
- Designing Traditional Cloning
- Cloning CRISPRi Knockdown System into Zymomonas mobilis
LCMS Protocols
- Lipid extraction, fatty acid methyl esterification (FAME), and GC-FID analysis
- Intracellular Metabolite Extraction
- Lipid and Fatty Acid Extraction Protocol
- Protocol for using E. coli as an internal standard for intracellular metabolite quantitation
- Aniline derivatization of metabolite samples
- Benzyl chloroformate derivatization of metabolite samples
- O-benzylhydroxylamine (O-BHA) derivatization of metabolite samples
- Preparing Solvent 1 with TBA (aka Solvent A; SolA)
- Lipid and Fatty Acid Extraction Protocol using Glass Pipettes
Lab Protocol Videos
- List of individual protocol videos
Software / Computer Protocols
- Connecting to the DAN lab server (Research Drive – Amador-Noguez)
- Running MZmine using scripts
- Quickstart guide for running INCA on the GLBRC servers
Proteomics Protocols
- Bradford Assay Protein Quantification
- Preparation of Cell Pellet for LCMS Proteomics or Protein Extraction
- Lysis of Cell Pellet for Protein Extraction Under Denaturing Conditions
- Protein Purification of His-tagged Proteins from Cell Lysate Under Denaturing Conditions
- SDS-PAGE Gel Protocol
- Preparation of Cell Pellet for LC-MS Based Proteomics
Data Processing/Analysis
- General metabolite data processing guide
- Labeled data analysis and processing