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
  
• 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.
• Phusion (pfu-sso7d) polymerase purification
  Draft: How to prepare and harvest pfu-sso7d polymerase.
  
• Keio and ASKA Collections
  Information about finding and using these E. coli strains.
• Find sources for chemicals
  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.
• Escherichia coli REL606 Genome Resources: Design Primers | Blast | Genbank | Browser |BioCyc
• Acinetobacter baylyi ADP1 Genome Resources: BLAST | BioCyc | Quick Reference
• Predicting transcription start sites in bacteria: BPROM | PPP | NNPP
• Notes about degenerate bases from IDT
• Ordering Primers | ICMB's IDT page
• Primer Database
• 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
• Illumina Next Generation Sequencing
  Protocol for NGS library generation

Genetic Modification and Cloning

• UV Mutagenesis to create a mutation library
  Make a library of mutants with UV irradiation.
• 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 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.
• 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.
• Megaprimer Megawhop Mutagenesis
  Simple and reliable method for introducing specific mutations into plasmids.

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.
• 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.
• Competence Assays
  Measure the transformation frequency in Acinetobacter baylyi.
• 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.

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
• R3C Assay

Accessing the Literature and Managing Citations

• 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
• Managing Citations Using Mendeley
  Keep track of manuscript references and generate their citations with ease

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