Table of Contents

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)

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