Barrick Lab :: Laboratory Protocols

Updating Protocols

• How to Create a Protocol
  Best practices for designing a protocol and for putting it on the lab Wiki.
• Updates Needed
  Request new protocols and changes to protocols.

Lab Organization

• Lab Notebooks
  Best practices for planning and recording your science.
• Strain Database
  How to search and enter samples in the lab database.
• Labeling and Tech Cart Rules
  How to label containers and how to use the tech carts.

Lab Stocks and General Practices

• Autoclave Sterilization
• Cleaning Glassware
  Including Small Flasks, Large Flasks, Test Tubes, and Plating Beads.
• Media Recipes
  Solid and liquid media for bacterial growth.
• Antibiotics, Supplements, Non-canonical amino acids
  Stock solutions and working concentrations of antibiotics and media supplements.
• Reagent and Buffer Recipes
  How to prepare common stocks of chemical solutions used in lab.
• Plasmids
  Sequences and characteristics of plasmids commonly used in lab.
• Standard Microbiological Practices
  aka the approximately Ten Commandments of microbiology lab research.
• E. coli strains
  Characteristics of E. coli strains commonly used in the lab.
• Keio and ASKA Collections
  Information about finding and using these E. coli strains.
• Freezing Strains
  How to freeze and archive strain samples.
• Find strains and plasmids
  List of commonly used culture/plasmid collections.
• Find sources for chemicals
  Search by structure, identify chemical synonyms, and find commercial sources.

PCR and Sequencing

• Sequencing/Genotyping Primer Design
  Design primers to validate mutations in evolved genomes.
• Custom Primer Design
  Design primers to amplify specific genome fragments.
• Escherichia coli REL606 Genome Resources: Design Primers | Blast | Genbank | Browser |BioCyc
• Ordering Primers | ICMB's IDT page | Degenerate bases
• Resuspending Primers
• Standard PCR
  • Which polymerase is right for me?
  • Easy DpnI Digestion
• Overlap PCR
• Agarose Gel Electrophoresis
• DNA Concentration Determination
  Protocols for Qubit, Nanodrop, and Agarose Gel DNA concentrations, and example data highlighting caveats for each.
• DNA Sequencing
• 16S rRNA Sequencing
  16S rRNA PCR and sequencing to identify unknown microbes
• Tools: Blast2Seq (NCBI) | ORF Finder (NCBI) | Translate (EMBOSS)
• MEGAWHOP
  Simple and reliable method for introducing specific mutations into plasmids.

Genetic Modification and Cloning

• Site Directed Mutagenesis
  QuickChange-based protocol for site directed mutagenesis in plasmids.
• Transformation:
  • Electrocompetent Cells
  • Chemically Competent Cells
  • Conjugation
  • Assessing Transformation Efficiency
• UV Mutagenesis
  Make a library of bacterial mutants with UV irradiation.
• Selecting Arabinose Marker Revertants
  Direct selection of Ara+ revertants from Ara– strains.
• Colony Transformation
  Rapid chemical transformation of E. coli colonies with high copy plasmids..
• Restriction Enzyme Cloning
  Insert a target DNA sequence into a plasmid via restriction digest and ligation.
• Gene Gorging Neutral Markers
  λ red genetic modification for mutations with a selectable or screenable marker using donor plasmid. For example, converting Ara+ strains to Ara–.
• Gene Gorging Evolved Alleles
  λ red genetic modification without a selectable or screenable phenotype using donor plasmid.
• FLP-FRT Recombination
  Induce targeted recombination (commonly used to eliminate the Kan^R cassette from Keio strains).
• Genome Modification (Datsenko and Wanner Method)
  λ red genetic modification using a PCR product containing a selectable marker.
• Gene Replacements (Church Method)
  Allelic exchange using pKOV integration. Generate unmarked mutations.
• Gibson Cloning
  Make complex constructs from multiple genetic parts. Master mix recipe, procedure, and Gibson walkthrough included.
• lambda red mediated ssDNA gene modification
  λ red mediated ssDNA oligo mutation reconstruction.
• pSLTS genomic editing for E. coli (Copley lab)
  Scarless genome modification using pSLTS plasmids developed by Copley lab.
• Golden Gate Assembly Protocols
  Design and build constructs that are compatible with Golden Gate Assembly
• P1 Transduction
  Quickly move mutations that are linked to a selectable marker between E. coli strains
• CRISPR Associated Transposons (CASTs)
  Targeted insertion of genetic cargos into bacterial genomes

Mutation Rates, Growth Rates and Evolutionary Stability

• Fluctuation Tests
  Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy.
• Growth Rates
  Measure bacterial growth from OD600/platereader data and calculate growth rates with the Growthcurver R package.
  • Growth Rates with Grofit
    Calculate growth rates with the Grofit R package.
• Plasmid Copy Number
  Measure copy number of plasmids compared to genome.
• Fluorescent Protein Evolutionary Stability
  Measure the evolutionary stability of fluorescent protein expression.
• CFU Counts
  Measure the number of CFU in a sample with this simple spot-plating method

Phage

• Making Phage Lysates
  Procedure for E. coli phages T4, T5, T6, and T7.
• Measuring Phage Titers
  Determine how many plaque-forming units are in a lysate.
• Isolating Phage Genomic DNA
  Purify DNA from a phage sample for sequencing or use as a PCR template.
• T7 Phage Fitness Assays:
  • Measuring Adsorption Rate of T7 Phage
  • Measuring Lysis Timing of T7 Phage
  • Measuring Burst Size of T7 Phage

Genome Resequencing and Mutation Validation

• Targeted Sequencing of a Specific Gene
  To find mutations or validate predictions from whole-genome re-sequencing data.
• Mutation Rates from Genome Resequencing
  How to estimate mutation rates (and confidence limits) from counts of mutations in multiple genome data sets.
• Next-generation Sequencing Illumina library preparation
  Steps for generating Illumina libraries.
• NGS Plasmid Sequencing
  sample submission requirements and process overview for sequencing plasmids on an iSeq.

Experimental Evolution

• Evolution Experiments General concerns in designing evolution experiments.
• Competitive Fitness Assays
  Measure the head-to-head relative fitness of two strains in co-culture.
• Identifying Ribose Operon Deletions
• Mutation Accumulation for Microsatellite Strains
• Bacteria Mutation Accumulation Experiments
  Propagate bacteria through single-cell bottlenecks to measure mutation rates.
• Selection in Emulsions
  Using emulsions to select by yield

Microplate Reader, Flow Cytometry, and Cell Sorting

• Microplate Reader Quick Start Guide
  Quick guide with main steps for setting up and using the microplate reader in our lab.
• Generic Flow Cytometry guidelines
  Guidelines for flow cytometry on the Fortessa (basic) instrument.
• Sorting Bacterial cells using the FACSAria
• Guidelines for sorting bacteria using fluorescence-activated cell sorting (FACS)

Nucleic Acid Preparation, RNA-Seq, qPCR

• Ethanol Precipitation
  Precipitate DNA to remove salts
• Phenol/chloroform bacterial genome extraction
  Protocol for chemical extraction of very pure bacterial DNA
• Bacterial total RNA isolation
  Preparation of high quality RNA using RNA Snap method combined with Zymo column purification.
• Bacterial dsRNA isolation
  Crude preparation of double-stranded RNA (dsRNA) using salt (NaCl).
• Plant RNA isolation
• RNAseq
  Preparation of rRNA-free libraries from bacterial culture for submission to GSAF core.
• qPCR Hub
  • Comparative RT-qPCR for differential gene expression
  • Absolute qPCR for plasmid copy number
• Denaturing formaldehyde gels
  Quick gels to verify size of RNA from e.g. transcriptions. Not suitable for purification.
• Polyacrylamide Gel Electrophoresis
  Denaturing and nondenaturing (native) polyacrylamide gel electrophoresis.
• In vitro Transcription

Protein Expression and Purification

• Large-scale Protein Expression in E. Coli
• Running SDS-PAGE Protein Gels
• Purifcation of 6xHis-Tagged Proteins from E. Coli lysates
• Pfu-Sso7d polymerase purification
  How to express and purify Pfu-Sso7d polymerase.

in vitro Enzyme Activity Assays

• Measuring transcription in vitro using fluorescent RNA aptamers (Broccoli, Spinach, etc.)
• Intracellular ROS detection

Working with Unusual Microbes

• Bees: Snodgrassella alvi | Gilliamella apicola | Bartonella apis | Lactobacillus "Firm-5"
• Aphids: Serratia symbiotica | Arsenophonus nasoniae
• Plants: Pseudomonas syringae
• Other: Vibrio natriegens (Vmax) | Serratia marcescens
• Yeast Saccharomyces cerevisiae

Acinetobacter baylyi ADP1

• Acinetobacter baylyi ADP1 Overview
  Useful information and links for working with this highly transformable bacterium.
• Competence Assays
  How to confirm competence and measure the transformation frequency in Acinetobacter baylyi.
• Transforming Acinetobacter baylyi ADP1
  How to transform Acinetobacter baylyi ADP1 with plasmids or PCR products.
  • Acinetobacter baylyi ADP1 "in plate" and "puddle" transformations
    Transformation of ADP1 in solid medium is known to achieve higher transformation efficiency. Quick and ideal for difficult transformations or when low amounts of transforming DNA are available.
• Acinetobacter baylyi ADP1 genome manipulation
  How to make deletions, insertions, and other modifications to the Acinetobacter baylyi genome.
  • ADP1 "Golden Transformation" genome manipulation
    How to make fast near scarless deletions to the Acinetobacter baylyi genome using a modified Golden Gate Cloning methodology.
  • ADP1 Overlap Extension PCR genome manipulation
    How to make modifications to the Acinetobacter baylyi genome with overlap PCRs.
• Quick 3hr Antibiotic Rescue Verification
  Same-day verification of antibiotic removal/"rescue" from counter-selection plates (e.g., AZT)
• ADP1 Tn-seq Library Prep
  Protocol for preparing a Tn-seq library for ADP1 strains mutagenized with pBT20

Working with Insects

• Aphid Care and Protocols
• Leafhopper Care and Protocols
• Tobacco hornworm (Manduca sexta) care

Useful Resources

• Retired and rarely used protocols
• Links to Product Manuals
  
• OpenWetWare a Wiki of biological protocols
• Enzymes used in molecular biology: a useful guide