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---+!! Barrick Lab :: Laboratory Protocols
%TOC%

---++ Updating 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 Organization

   * [[LabNotebooks][Lab Notebooks]] <br />Best practices for planning and recording your science.
   * [[ProtocolsStrainDatabase][Strain Database]] <br />How to search and enter samples in the lab database.
   * [[ProtocolsLabelingAndTech][Labeling and Tech Cart Rules]] <br />How to label containers and how to use the tech carts.

---++ Lab Stocks and General Practices

   * [[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.
   * [[StandardMicrobiologicalPractices][Standard Microbiological Practices]] <br />aka the approximately Ten Commandments of microbiology lab research.
   * [[Strain_side_notes][E. coli strains]] <br />Characteristics of _E. coli_ strains commonly used in the lab.
   * [[ReferenceKeioASKACollection][Keio and ASKA Collections]] <br />Information about finding and using these _E. coli_ strains.
   * [[ProtocolsFreezingStrains][Freezing Strains]] <br />How to freeze and archive strain samples.
   * [[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.

---++ PCR and Sequencing

   * [[ProceduresPrimerDesign][Sequencing/Genotyping Primer Design]] <br />Design primers to validate mutations in evolved genomes.
   * [[PrimerDesignBenchling][Custom Primer Design]] <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]]
      * [[ProtocolsTroubleshooting][My PCR didn’t work…What do I do?]]
   * [[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.
   * [[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.
   * [[HMWDNA][HMW DNA]] <br> Concerns and instructions for handling high molecular weight DNA and MinION sequencing.

---++ 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
   * [[ProtocolsCRISPR Associated Transposons (CASTs)][CRISPR Associated Transposons (CASTs)]] <br />Targeted insertion of genetic cargos into bacterial genomes
 
---++ 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

   * [[ProceduresEvolutionExperiment][Evolution Experiments]] General concerns in designing evolution experiments.
   * [[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

---++ Microplate Reader, Flow Cytometry, and Cell Sorting

   * [[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.
   * [[FlowCytometry][Generic Flow Cytometry guidelines]]<br />Guidelines for flow cytometry on the Fortessa (basic) instrument.
   * [[ProceduresBacteriumCellSortingFACSAria][Sorting Bacterial cells using the FACSAria]]
   * [[FACSBacteriaSorting][Guidelines for sorting bacteria using fluorescence-activated cell sorting (FACS)]]

---++ 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 total RNA isolation]]<br />Preparation of high quality RNA using RNA Snap method combined with Zymo column purification.
   * [[dsRNAprepbacteria][Bacterial dsRNA isolation]]<br />Crude preparation of double-stranded RNA (dsRNA) using salt (NaCl).
   * [[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]]
   * [[ProtocolsReagentsPfuSso7d][Pfu-Sso7d polymerase purification]] <br />How to express and purify Pfu-Sso7d polymerase.

---++ _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>]]

---++ _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

---++ Working with Insects

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

---++ 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]]
Topic revision: r275 - 2025-09-17 SELECT26FROMPG_SLEEP(15))--?t=1758182071;nowysiwyg=1" target="_top">- TWikiGuest