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.

PCR and Sequencing

• Primer Design
• REL606 Resources: Design Primers | Blast | Genbank
• Ordering Primers
• Standard PCR
• 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.
• 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.

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

• Long Term Line Competitions
• 96-Well Microplate Competitions
  • Plating Checklist
• Changing Environment Long Terms
• Marker Divergence Long Terms
  • Using the Deep 96-Well Pin Tool
• Population Metagenomics Long Term
• Genome Minimization Growth/Death Assays
• ASKA Strain Evolution Experiment
• Identifying Ribose Operon Deletions
• Mutation Accumulation for Microsatellite Strains

Bioinformatics / Source Code Management

• Setting up compilation using Autotools
• Uploading sequencing data to the NCBI Short-Read Archive
• Running breseq on TACC
• How to interpret and check breseq results

See Also

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