Barrick Lab :: 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
  Sequences and characteristics of plasmids commonly used in lab.
• E. coli Genotypes (OWW)
  Common genotypes, genetic markers, and alleles.
• 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.
• Keio and ASKA Collections
  Information about finding and using these E. coli strains.
• Find chemicals using SciFinder
  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.
• REL606 Genome Resources: Design Primers | Blast | Genbank | Browser |BioCyc
• Predicting transcription start sites in bacteria: BPROM | PPP | NNPP
• Ordering Primers | ICMB's IDT page
• Primer Database
• Resuspending Primers
• Standard PCR
  • Which polymerase is right for me?
• Overlap PCR
• Agarose 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)
• MEGAWHOP

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.
• FLP-FRT Recombination
  Induce targeted recombination (commonly used to eliminate the Kan^R cassette 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

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 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.
• Pulsed-Field Gel Electrophoresis (PFGE) Mapping
  To validate large-scale chromosomal rearrangements, deletions, and duplications.
• Mutation Rates from Genome Resequencing
  How to estimate mutation rates (and confidence limits) from counts of mutations in multiple genome data sets.
• Generic Flow Cytometry guidelines
  Guidelines for flow cytometry on the fortessa (basic) instrument.

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.

Nucleic Acid In Vitro Selection

• Spectrophotometric Nucleic Acid Quantitation
  Determine concentrations of oligonucleotides and other nucleic acids from absorption spectra.
• Polyacrylamide Gel Electrophoresis
  Denaturing and nondenaturing (native) polyacrylamide gel electrophoresis.
• Crush-Soak Elution
  Recover DNA or RNA samples from polyacrylamide gels.
• Ethanol Precipitation
  Precipitate DNA or RNA samples to remove salts and concentrate.
• Primer Extension
• PCR for Nucleic Acid Selections
• Total Alkaline Digest of Embedded RNA linkages
• List Primers Deoxyribozymes

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 and Presenting

• Barrick Lab Style Guide
• How to Graph Data
• Subtle Grammatical Usage Rules
• Science Quotations

See Also

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

Misc. Lab Protocols

• Autoclave Use
• Cleaning Glassware
• Reusing plating beads
• Measuring the Rate of Attrition in frozen or cooled samples