Protocols and Laboratory Reference

Stock Solutions

• Reagent and Buffer Recipes
• Media Recipes
• Antibiotic Stock Solutions and Working Concentrations

PCR

• Primer Design
• Design Primers for REL606 (NCBI)
• Ordering Primers
• Standard PCR
• 16S rRNA PCR and Sequencing to Identify Unknown Microbes

Sequence Analysis

• REL606 Resources: Blast Genbank
• Blast2Seq (NCBI)
• ORF Finder (NCBI)
• EMBOSS Transeq - DNA to protein translation.

Microbiology

• Fluctuation Tests
  Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy.
• Phage T5 Fluctuation Test
• Phage Lysate Preparation (T4,T5,T6) and Determining Phage Titer
• Making Electrocompetent Cells
• UV Mutagenesis
• E. coli Microarray Experiments
• Commonly used plasmids
• Cloning and Strain Construction Projects

Genetic Modification

• Gene Gorging Neutral Markers
• Gene Gorging Evolved Alleles
• Gene Knockouts (Datsenko and Wanner Method) (Keio Strain Background)

Genome Re-sequencing and Mutation Validation

• Genomic DNA Isolation
  Isolate gDNA for genome re-sequencing.
• Targeted Sequencing of a Specific Gene
  To validate point mutations and small indels predicted from whole-genome re-sequencing.
• Pulsed-Field Gel Electrophoresis (PFGE) Mapping
  To validate large-scale chromosomal rearrangements, deletions, and duplications.
• Veracode Golden Gate Genotyping

Experimental Evolution of Bacteria

• Selecting Arabinose Marker Revertants
• 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

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