(r261) Barrick Lab :: Laboratory Protocols

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---+!! Barrick Lab :: Laboratory Protocols
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---++ Creating Protocols
   * [[ProtocolHowTo][How to Create a Protocol]] %BR% Best practices for designing a protocol and for putting it on the lab Wiki.
   * [[ProtocolUpdatesNeeded][Updates Needed]] %BR% Request new protocols and changes to protocols.

---++ Lab Stocks and Practices
   * [[LabNotebooks][Lab Notebooks]] <br />Best practices for planning and recording your science.
   * [[StandardMicrobiologicalPractices][Standard Microbiological Practices]] <br />aka the approximately Ten Commandments of microbiology lab research.
   * [[AutoclaveSterilization][Autoclave Sterilization]]
   * [[GlasswareCleaning][Cleaning Glassware]] <br />Including Small Flasks, Large Flasks, Test Tubes, and Plating Beads.
   * [[ProtocolsMediaRecipes][Media Recipes]] <br />Solid and liquid media for bacterial growth.
   * [[ProtocolsAntibioticStockSolutions][Antibiotics, Supplements, Non-canonical amino acids]] <br />Stock solutions and working concentrations of antibiotics and media supplements.
   * [[ProtocolsReagentRecipes][Reagent and Buffer Recipes]] <br />How to prepare common stocks of chemical solutions used in lab.
   * [[ReferencePlasmids][Plasmids]] <br />Sequences and characteristics of plasmids commonly used in lab.
   * [[ProtocolsFreezingStrains][Freezing Strains]] <br />How to freeze and archive strain samples.
   * [[ProtocolsStrainDatabase][Strain Database]] <br />How to search and enter samples in the lab database.
   * [[ProtocolsReagentsPfuSso7d][Pfu-Sso7d polymerase purification]] <br />How to express and purify Pfu-Sso7d polymerase.
   * [[ReferenceKeioASKACollection][Keio and ASKA Collections]] <br />Information about finding and using these _E. coli_ strains.
   * [[ProtocolsFindStrainsPlasmids][Find strains and plasmids]] <br />List of commonly used culture/plasmid collections.
   * [[ProtocolsFindChemicals][Find sources for chemicals]] <br />Search by structure, identify chemical synonyms, and find commercial sources.
   * [[Strain_side_notes][E. coli strains]] <br />Characteristics of _E. coli_ strains commonly used in the lab.
   * [[Microplate Reader Quick Start Guide][Microplate Reader Quick Start Guide]] <br />Quick guide with main steps for setting up and using the microplate reader in our lab.
  

---++ PCR and Sequencing

   * [[ProceduresPrimerDesign][Sequencing/Genotyping Primer Design]] <br />Design primers to validate mutations in evolved genomes.
   * [[PrimerDesignBenchling][Custom Primer Design on Benchling]] <br />Design primers to amplify specific genome fragments.
   * _Escherichia coli_ REL606 Genome Resources: [[http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=413997&INPUT_SEQUENCE=NC_012967.1&log$=seqview_list_primer][Design Primers]] | [[http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&USER_FORMAT_DEFAULTS=on&SET_SAVED_SEARCH=true&PAGE=MegaBlast&PROGRAM=blastn&GAPCOSTS=0%200&MATCH_SCORES=1,-2&DATABASE=nr&BLAST_PROGRAMS=megaBlast&MAX_NUM_SEQ=100&SHORT_QUERY_ADJUST=on&EXPECT=10&WORD_SIZE=28&REPEATS=repeat_9606&TEMPLATE_TYPE=0&TEMPLATE_LENGTH=0&FILTER=L&FILTER=m&EQ_MENU=Escherichia%20coli%20B%20str.%20REL606%20%28taxid%3A413997%29&SHOW_OVERVIEW=on&SHOW_LINKOUT=on&ALIGNMENT_VIEW=Pairwise&MASK_CHAR=2&MASK_COLOR=1&GET_SEQUENCE=on&NUM_OVERVIEW=100&DESCRIPTIONS=100&ALIGNMENTS=100&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&OLD_BLAST=false][Blast]] | [[http://www.ncbi.nlm.nih.gov/nuccore/254160123][Genbank]] | [[http://www.ncbi.nlm.nih.gov/nuccore/NC_012967.1?report=graph][Browser]] |[[http://www.ecocyc.org/ECOL413997/][BioCyc]]
   * [[ProtocolsOrderingPrimers][Ordering Primers]] | [[http://www.idtdna.com/ICMBSupplyCenter/Login.aspx][ICMB's IDT page]] %ICON{external}% | [[ProtocolsDegenerateBases][Degenerate bases]]
   * [[ProtocolsResuspendingPrimers][Resuspending Primers]]
   * [[ProtocolsStandardPCR][Standard PCR]] 
      * [[ProtocolsTaq][Which polymerase is right for me?]]
      * [[ProtocolsDpnIDigestion][Easy DpnI Digestion]]
   * [[ProceduresOverlapPCR][Overlap PCR]]
   * [[ProceduresStandardAgaroseGel][Agarose Gel Electrophoresis]]
   * [[DNAConcentrationDetermination][DNA Concentration Determination]]<br />Protocols for Qubit, Nanodrop, and Agarose Gel DNA concentrations, and example data highlighting caveats for each.
   * [[ProcedureDNASequencing][DNA Sequencing]]
   * [[Protocols16SSequencing][16S rRNA Sequencing]]<br />16S rRNA PCR and sequencing to identify unknown microbes
   * Tools: [[http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi][Blast2Seq (NCBI)]] | [[http://www.ncbi.nlm.nih.gov/gorf/gorf.html][ORF Finder (NCBI)]] | [[http://www.ebi.ac.uk/Tools/emboss/transeq/index.html][Translate (EMBOSS)]]
   * [[MEGAWHOP][MEGAWHOP]] <br> Simple and reliable method for introducing specific mutations into plasmids.

---++ Genetic Modification and Cloning
   * [[ProtocolsQuickChange][Site Directed Mutagenesis]] %BR% QuickChange-based protocol for site directed mutagenesis in plasmids.
   * Transformation:  
      * [[ProtocolsElectrocompetentCells][Electrocompetent Cells]]
      * [[ProtocolsChemCompCellsHub][Chemically Competent Cells]]
      * [[ProtocolsConjugation][Conjugation]]
      * [[ProtocolsTestingTransformEff][Assessing Transformation Efficiency]]
   * [[ProtocolsUVLibrary][UV Mutagenesis]] %BR% Make a library of bacterial mutants with UV irradiation.
   * [[ProtocolsAraMarker][Selecting Arabinose Marker Revertants]] %BR% Direct selection of Ara+ revertants from Ara&ndash; strains.
   * [[ProtocolsColonyTransformation][Colony Transformation]] %BR% Rapid chemical transformation of E. coli colonies with high copy plasmids..
   * [[ProtocolsRestrictionEnzymeCloning][Restriction Enzyme Cloning]] %BR% Insert a target DNA sequence into a plasmid via restriction digest and ligation.
   * [[ProtocolsGeneGorgingMarker][Gene Gorging Neutral Markers]] %BR% &lambda; red genetic modification for mutations with a selectable or screenable marker using donor plasmid. For example, converting Ara+ strains to Ara&ndash;.
   * [[ProceduresGeneGorgingEvolvedAlleles][Gene Gorging Evolved Alleles]] %BR% &lambda; red genetic modification without a selectable or screenable phenotype using donor plasmid.
   * [[ProcedureFLPFRTRecombination][FLP-FRT Recombination]] %BR% Induce targeted recombination (commonly used to eliminate the Kan<sup>R</sup> cassette from Keio strains).
   * [[ProcedureGenomeModificationDatsenkoWanner][Genome Modification (Datsenko and Wanner Method)]] %BR% &lambda; red genetic modification using a PCR product containing a selectable marker.
   * [[ProcedureGeneReplacements][Gene Replacements (Church Method)]]%BR% Allelic exchange using pKOV integration. Generate unmarked mutations.
   * [[ProtocolsGibsonCloning][Gibson Cloning]] %BR% Make complex constructs from multiple genetic parts. Master mix recipe, procedure, and Gibson walkthrough included.
   * [[ProtocolsSsdnaRecombination][lambda red mediated ssDNA gene modification]]<br />&lambda; red mediated ssDNA oligo mutation reconstruction.
   * [[ProtocolsPsltsEditing][pSLTS genomic editing for _E. coli_ (Copley lab)]]<br />Scarless genome modification using pSLTS plasmids developed by Copley lab.
   * [[Golden Gate Assembly Protocols Main Page][Golden Gate Assembly Protocols]]<br/>Design and build constructs that are compatible with Golden Gate Assembly
   * [[ProtocolsP1Transduction][P1 Transduction]] <br />Quickly move mutations that are linked to a selectable marker between _E. coli_ strains

 
---++ Mutation Rates, Growth Rates and Evolutionary Stability

   * [[ProtocolsFluctuationTests][Fluctuation Tests]]<br />Measure bacterial mutation rates to antibiotic resistance, phage resistance, or reversion of auxotrophy.
   * [[ProtocolsGrowthRates][Growth Rates]]<br />Measure bacterial growth from OD600/platereader data and calculate growth rates with the Growthcurver R package.
      * [[ProtocolGrofit][Growth Rates with Grofit]]<br />Calculate growth rates with the Grofit R package.
   * [[ProtocolsPlasmidCopyNumber][Plasmid Copy Number]]<br />Measure copy number of plasmids compared to genome.
   * [[ProtocolsFluorescentProteinEvolutionaryStability][Fluorescent Protein Evolutionary Stability]]<br />Measure the evolutionary stability of fluorescent protein expression.
   * [[ProtocolsCFUCounts][CFU Counts]]<br />Measure the number of CFU in a sample with this simple spot-plating method

---++ Phage

   * [[ProtocolPhageLysate][Making Phage Lysates]]%BR%Procedure for _E. coli_ phages T4, T5, T6, and T7.
   * [[ProtocolsPhageTiters][Measuring Phage Titers]]%BR%Determine how many plaque-forming units are in a lysate.
   * [[ProtocolsPhageGenomicDNA][Isolating Phage Genomic DNA]]%BR%Purify DNA from a phage sample for sequencing or use as a PCR template.
   * T7 Phage Fitness Assays: 
      * [[ProtocolsPhageAdsorptionRate][Measuring Adsorption Rate of T7 Phage]]
      * [[ProtocolsPhageLysisTiming][Measuring Lysis Timing of T7 Phage]]
      * [[ProtocolsPhageBurstSize][Measuring Burst Size of T7 Phage]]

---++ Genome Resequencing and Mutation Validation

   * [[ProceduresTargetedSequencing][Targeted Sequencing of a Specific Gene]]<br />To find mutations or validate predictions from whole-genome re-sequencing data.
   * [[ProceduresCalculatingMutationRatesFromGenomicData][Mutation Rates from Genome Resequencing]] %BR% How to estimate mutation rates (and confidence limits) from counts of mutations in multiple genome data sets.
   * [[ProceduresKappaNgsPrep][Next-generation Sequencing Illumina library preparation]] %BR% Steps for generating Illumina libraries.
   * [[ProceduresPlasmidSequencing][NGS Plasmid Sequencing]] %BR% sample submission requirements and process overview for sequencing plasmids on an iSeq.

---++ Experimental Evolution

   * [[ProceduresLongTermCompetitions][Competitive Fitness Assays]] <br />Measure the head-to-head relative fitness of two strains in co-culture.
   * [[ProceduresEvolvabilityRiboseSubpopulations][Identifying Ribose Operon Deletions]]
   * [[ProcedureEvolvabilityMutationAccumulation][Mutation Accumulation for Microsatellite Strains]]
   * [[ProcedureBacterialMutationAccumulation][Bacteria Mutation Accumulation Experiments]] %BR% Propagate bacteria through single-cell bottlenecks to measure mutation rates.
   * [[ProcedureSelectingByEfficiency][Selection in Emulsions]] %BR% Using emulsions to select by yield

---++ Flow Cytometry and Cell Sorting

   * [[FlowCytometry][Generic Flow Cytometry guidelines]]<br />Guidelines for flow cytometry on the Fortessa (basic) instrument.
   * [[ProceduresBacteriumCellSortingFACSAria][Sorting Bacterial cells using the FACSAria]]

---++ Nucleic Acid Preparation, RNA-Seq, qPCR

   * [[ProtocolsEthanolPrecipitation][Ethanol Precipitation]] %BR% Precipitate DNA to remove salts
   * [[PhenolChloroformExtraction][Phenol/chloroform bacterial genome extraction]] %BR% Protocol for chemical extraction of very pure bacterial DNA
   * [[RNAPrep][Bacterial RNA isolation]]<br />Preparation of high quality RNA using RNA Snap method combined with Zymo column purification.
   * [[RNAPlantPrep][Plant RNA isolation]]
   * [[RNASeq][RNAseq]]<br />Preparation of rRNA-free libraries from bacterial culture for submission to GSAF core.
   * [[qPCR_hub][qPCR Hub]]
      * [[qPCR][Comparative RT-qPCR for differential gene expression]]
      * [[qPCR to quantify plasmid copy number][Absolute qPCR for plasmid copy number]]
   * [[Denaturing formaldehyde gels][Denaturing formaldehyde gels]]<br />Quick gels to verify size of RNA from e.g. transcriptions. Not suitable for purification.
   * [[ProceduresPolyacrylamideGelElectrophoresis][Polyacrylamide Gel Electrophoresis]] %BR% Denaturing and nondenaturing (native) polyacrylamide gel electrophoresis.
   * [[RNAT7][ _In vitro_ Transcription]]

---++ Protein Expression and Purification

   * [[ProtocolsLargeScaleProteinExpressionEColi][Large-scale Protein Expression in E. Coli]]
   * [[ProtocolsRunningSDSPAGEProteinGels][Running SDS-PAGE Protein Gels]]
   * [[Protocols6xHisTagProteinPurification][Purifcation of 6xHis-Tagged Proteins from E. Coli lysates]]

---++ _in vitro_ Enzyme Activity Assays

   * [[ProtocolsBroccoliAptamerInVitroAssays][Measuring transcription _in vitro_ using fluorescent RNA aptamers (Broccoli, Spinach, etc.)]]
   * [[ProtocolsIntrocellularROSDetection][Intracellular ROS detection]]

---++ Working with Unusual Microbes
   * Bees: [[ProtocolsCulturingSnodgrassellaAlvi][<em>Snodgrassella alvi</em>]] | [[ProtocolsCulturingGilliamellaApicola][Gilliamella apicola]] | [[ProtocolsCulturingBartonellaApis][<em>Bartonella apis</em>]] | [[ProtocolsCulturingFirm_5][<em>Lactobacillus</em> "Firm-5"]]
   * Aphids: [[ProtocolsWorkingWithSerratiaSymbiotica][<em>Serratia symbiotica</em>]] | [[ProtocolsCulturingArsenophonusNasoniae][<em>Arsenophonus nasoniae</em>]]
   * Plants: [[ProtocolsWorkingWithPseudomonasSyringae][<em>Pseudomonas syringae</em>]]
   * Other: [[ProtocolsWorkingWithVibrioNatriegens][<em>Vibrio natriegens</em> (Vmax)]] | [[ProtocolsWorkingWithSerratiaMarcescens][<em>Serratia marcescens</em>]]
   * Yeast [[ProtocolsLithiumAcetateTransformation][<i>Saccharomyces cerevisiae</i>]]

---++ Working with Insects

   * [[ProtocolsAphids][Aphid Care and Protocols]]
   * [[ProtocolsLeafhoppers][Leafhopper Care and Protocols]]
   * [[ProtocolsHornwormCare][Tobacco hornworm (<em>Manduca sexta</em>) care]]

---++ _Acinetobacter baylyi_ ADP1

   * [[ProtocolsAcinetobacterBaylyiADP1][ _Acinetobacter baylyi_ ADP1 Overview]] %BR% Useful information and links for working with this highly transformable bacterium.
   * [[ProceduresCompetenceAssays][Competence Assays]] <br />How to confirm competence and measure the transformation frequency in _Acinetobacter baylyi_.
   * [[ProtocolsAcinetobacterTransformation][Transforming <em>Acinetobacter baylyi</em> ADP1]]<br />How to transform _Acinetobacter baylyi_ ADP1 with plasmids or PCR products.
      * [[ProtocolsAcinetobacterPuddleTransformation][<em>Acinetobacter baylyi</em> ADP1 "in plate" and "puddle" transformations]]<br />Transformation of ADP1 in solid medium is known to achieve higher transformation efficiency. Quick and ideal for difficult transformations or when low amounts of transforming DNA are available.
   * [[ProtocolsAcinetobacterGenomeManipulation][<em>Acinetobacter baylyi</em> ADP1 genome manipulation]]<br />How to make deletions, insertions, and other modifications to the _Acinetobacter baylyi_ genome.
      * [[ProtocolsAcinetobacterGoldenTransformation][ADP1 "Golden Transformation" genome manipulation]]<br />How to make fast near scarless deletions to the _Acinetobacter baylyi_ genome using a modified Golden Gate Cloning methodology.
      * [[ProtocolsAcinetobacterOverlapPCRTransformation][ADP1 Overlap Extension PCR genome manipulation]]<br />How to make modifications to the _Acinetobacter baylyi_ genome with overlap PCRs.
   * [[ProtocolsAntibioticRemovalVerification][Quick 3hr Antibiotic Rescue Verification]]<br/>Same-day verification of antibiotic removal/"rescue" from counter-selection plates (e.g., AZT)
   * [[ADP1 Tn-seq Library Prep][ADP1 Tn-seq Library Prep]]<br />Protocol for preparing a Tn-seq library for ADP1 strains mutagenized with pBT20

---++ Useful Resources
   * %ICON{airplane}% [[ProtocolsRetired][Retired and rarely used protocols]]
   * %ICON{help}% [[ProductManualLinks][Links to Product Manuals]]%BR%
   * [[http://openwetware.org/wiki/Electrocompetent_cells][OpenWetWare]] a Wiki of biological protocols
   * [[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570007/][Enzymes used in molecular biology: a useful guide]]
Barrick Lab > ProtocolList
Contributors to this topic
JeffreyBarrick, SeanLeonard, DanielDeatherage, PengGeng, CraigBarnhart, ColinBrown, KateElston, GabrielSuarez, AurkoDasgupta, SimonDAlton, IsaacGifford, BrianRenda, LindseyWolf, MichaelHammerling, MattMcGuffie, JordanMonk, DaciaLeon, ElizabethRobinson, AlvaroRodriguez, JuliePerreau, MarkKauth, JaredEller, SophiaLin, DennisMishler, StrattonGeorgoulis, LucioNavarro, SarahBialik, SteveSowa, XueZhang, CamiloGomez, NeilGottel, WaldoDittmar, AnthonyVandieren, EricBryan
Topic revision: r261 - 2023-02-23 - 17:48:13 - Main.IsaacGifford