Barrick Lab :: Laboratory Protocols

Creating 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 Stocks and Practices

• Standard Microbiological Practices
  aka the approximately Ten Commandments of microbiology lab research.
• Autoclave Sterilization
• Cleaning Glassware
  Including Small Flasks, Large Flasks, Test Tubes, and Plating Beads.
• Media Recipes
  Solid and liquid media for bacterial growth.
• Antibiotics and Supplements
  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.
• Freezing Strains
  How to freeze and archive strain samples.
• Strain Database
  How to search and enter samples in the lab database.
• Pfu-Sso7d polymerase purification
  How to express and purify Pfu-Sso7d polymerase.
• Keio and ASKA Collections
  Information about finding and using these E. coli strains.
• 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.
• E. coli strains
  Characteristics of E. coli strains commonly used in the lab.
• Microplate Reader Quick Start Guide
  Quick guide with main steps for setting up and using the microplate reader in our lab.
• How We Send Strains by Mail
  How to handle strain requests and send by mail.

PCR and Sequencing

• Sequencing/Genotyping Primer Design
  Design primers to validate mutations in evolved genomes.
• 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

Genetic Modification and Cloning

• Conjugation
  General conjugation protocol for introducing plasmids to bacteria.
• UV Mutagenesis
  Make a library of bacterial mutants with UV irradiation.
• Selecting Arabinose Marker Revertants
  Direct selection of Ara+ revertants from Ara– strains.
• Electrocompetent Cells
  Make electrocompetent E. coli stocks for transformation.
• Electroporation of your Electrocompetent Cells
  Transform your Electrocompetent cells with DNA.
• 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.
• Megaprimer Megawhop Mutagenesis
  Simple and reliable method for introducing specific mutations into plasmids.
• Golden Gate Assembly Protocols
• Bee Microbiome Toolkit (BTK)
  Golden Gate part kit for work in non-model bacterial species
• P1 Transduction
  Quickly move mutations that are linked to a selectable marker between E. coli strains
• Preparing and Transforming Chemically Competent Cells
  No-kit Inoue method
  • Transforming Chemically Competent Cells
  • Checking Transformation Efficiency of Chemically Competent Cells
• Preparing Chemically Competent Cells -- quick and efficient
  CaCl₂/Glycerol method

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 rates from OD600/platereader data
• Fluorescent Protein Evolutionary Stability
  Measure the evolutionary stability of fluorescent protein expression.
• Making Phage Lysates
  E. coli phages T7, T4, others?
• Measuring Phage Titers
• 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

• Genomic DNA Isolation
  Isolate gDNA for genome resequencing.
• 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.

Experimental Evolution

• 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

Flow Cytometry and Cell Sorting

• Generic Flow Cytometry guidelines
  Guidelines for flow cytometry on the Fortessa (basic) instrument.
• Sorting Bacterial cells using the FACSAria

Nucleic Acid Preparation, RNA-Seq, qPCR

• Ethanol Precipitation
  Precipitate DNA to remove salts
• Bacterial RNA isolation
  Preparation of high quality RNA using RNA Snap method combined with Zymo column purification.
• Plant RNA isolation
• RNAseq
  Preparation of rRNA-free libraries from bacterial culture for submission to GSAF core.
• RT-qPCR for differential gene expression
• 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.

Non-canonical Amino Acids

• Working with an Expanded Genetic Code
  Properties of amino acids—both canonical and non-canonical—and their use in organisms with an expanded genetic code.
• Preparing Stock Solutions for ncAA
  Making stock solutions for non-canonical amino acids (ncAA).

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

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 | Serratia marcescens

Acinetobacter baylyi ADP1

• Genome Resources: BLAST | BioCyc | Quick Reference
• Competence Assays
  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 genome manipulation
  How to make deletions, insertions, and other modifications to the Acinetobacter baylyi genome.
• Acinetobacter baylyi ADP1 "Golden Transformation" genome manipulation
  How to make fast near scarless deletions to the Acinetobacter baylyi genome using a modified Golden Gate Cloning methodology.
• 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.
• 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

Yeasty Protocols

• Lithium Acetate Transformation

Accessing the Literature and Managing Citations and presentation information

• Off Campus Journal Access
  Methods of finding, discovering and accessing various journal articles
• Saved Article Searches
  Set up automatic updates to find current publications of interest
• How to make a good presentation
  How to make a good presenation under different considerations.
• How to (legally) get a copy of inaccessible journal articles
  How to access journal articles where either (i.) UT doesn't have a subscription or (ii.) Article only exists as a physical copy

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