Protocols and Laboratory Reference

• Updates Needed

Lab Stocks

• 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
  Information about plasmids commonly used in lab.
• Strain Database
  How to search and enter samples in the lab database.
• Freezing Strains
  How to freeze and archive strain samples.
• Lab Stocks
  List of amount of common lab stock solutions/media to keep on hand.

PCR and Sequencing

• Sequencing/Genotyping Primer Design
  Design primers to validate mutations in evolved genomes.
• REL606 Genome Resources: Design Primers | Blast | Genbank | Browser |BioCyc
• Predicting transcription start sites in bacteria: BPROM | PPP | NNPP
• Ordering Primers
• Primer Database
• Resuspending Primers
• Standard PCR
  • Which polymerase is right for me?
• Overlap PCR
• Agarose Gel Electrophoresis
• Polyacrylamide Gel Electrophoresis
• DNA Sequencing
• Using APE
• 16S rRNA Sequencing
  16S rRNA PCR and Sequencing to Identify Unknown Microbes
• Tools: Blast2Seq (NCBI) | ORF Finder (NCBI) | Translate (EMBOSS)

Genetic Modification

• UV Mutagenesis
• Selecting Arabinose Marker Revertants
  Direct selection of Ara+ revertants from Ara– strains.
• Electrocompetent Cells
  Make electrocompetent cell stocks for transformation.
• Ethanol Precipitation
  Precipitate DNA to remove salts before electroporation.
• 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.
• Gene Knockouts (Datsenko and Wanner Method)
  λ red genetic modification with a selectable marker using PCR product.
• Gene Replacements (Church Method)
  Generating precise indels/SNPs using pKOV integration.
• Gibson Cloning

Mutation Rates

• Fluctuation Tests
  Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy.
• Phage Stocks
  Phage Lysate Preparation (T4,T5,T6) and Determining Phage Titer

Genome Re-sequencing and Mutation Validation

• Genomic DNA Isolation
  Isolate gDNA for genome re-sequencing.
• Targeted Sequencing of a Specific Gene
  To find mutations or validate predictions from whole-genome re-sequencing data.
• Pulsed-Field Gel Electrophoresis (PFGE) Mapping
  To validate large-scale chromosomal rearrangements, deletions, and duplications.

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.

Bioinformatics / Source Code Management

• Analyzing RNA-Seq data
  Analyzing RNA-Seq data for differential expression.
• Setting up compilation using Autotools
• Sequence Read Archive Upload
  Uploading sequence data and obtaining an accession number for sequencing data from the SRA database.
• Running breseq on TACC
• Interpreting breseq output.
  How to interpret, check, and manually correct breseq results.

Environmental Microbiology

• Extracting Clean Metagenomic DNA From Soil

Writing

• Barrick Lab Style Guide

See Also

• OpenWetWare a Wiki of biological protocols
• Retired and rarely used protocols

Misc. Lab Protocols

• Autoclave Use
• Cleaning Glassware
• Reusing plating beads