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8.1- Characterization & DNA amplification

8.1.1-PCR
8.1.1.1-General PCR Methods & Guidelines

8.1.1.1.3-Basic GoTaq PCR Protocol.pdf [check with current users; keep]

8.1.1.1.4-Colony PCR Protocol [updated 29Jul2016 jit7]

8.1.1.1.5-Guidelines for Primer Design [Updated by vg93 27Oct2016]

8.1.1.1.5.1-Primer Design Checklist [created by vg93 31Oct2016]

8.1.1.1.7-Primer _ Ordering .pdfand Reconstitution SOP [updated 2Sept2016 nrs13]8.1.1.1.8-Singe-tube Sequencing Submission to BRC Protocol [updated 2Aug2016 by ajh222020-06-01 ram524]

8.1.1.1.9-Ten Things That Can Kill Your PCR Information Sheet [reviewed by mw16 07072016]

...

8.1.1.1.11-Primer Database Search Protocol.pdf SOP [updated 2Sept2016 nrs132020-06-01 ram524]  

8.1.1.1.12-rpoB _sigB_ATassignment_User.pdfsigB AT Assignment [MW]

8.1.1.1.13-PCR and Sanger Sequencing Reactions in 96-Well Plates (High-Throughput PCR and Sequencing)17-pGEM Ladder for Size Determination of PCR Products SOP [reviewed by mw16 14Sept2015]8.1.1.1.14-rpoB PCR and Sequencing SOP [reviewed 14Sept2015 by mw16updated by ser15 2020-05-18]

8.1.1.1.1518-PCR Amplification of Full Length and Internal Region of 16s rDNA SOP [reviewed 14Sept2015 by mw16]

8.1.1.2-Listeria PCR

8.1.1.2.1-Ten Gene MLST for Listeria

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Gel Electrophoresis of PCR Product SOP [updated 2020-05-18 by ser15]

8.1.1

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.1.

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19-

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PCR Product Purification and Sanger Sequencing Submission SOP [updated 2020-05-18 by ser15]

8.1.1.

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1.

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8.1.1.2.1.5-lmo1555 PCR-GoTaq.xls [reviewed by mw16 07072016]

8.1.1.2.1.6-lmo2763 PCR-GoTaqhotstart.xls [reviewed by mw16 07072016]

8.1.1.2.1.7-pbpA PCR-GoTaqhotstart.xls [reviewed by mw16 07072016]

...

20-Sanger Sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit SOP [created 2021-01-14 by sjr267]

Sequencing Cost Comparison Supplemental File [created 2020-12-25 by sjr267]

8.1.1.2

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-

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Listeria PCR

8.1.1.2

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.

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8.1.1.2.1.11-rarA PCR-GoTaq.xls [reviewed by mw16 07072016]

8.1.1.2.2-Listeria PCRs by Gene Name

8.1.1.2.2.1-Fact Sheet on ActA Differentiation PCR and Interpretation of Results.pdf [check with Shelley or sherry, should be superceeded by SOP; archived?]8.1.1.2.2.2-Gap PCR protocol for Listeria monocytogenes PCR Protocol [reviewed by mw16 07072016actA PCR SOP [updated 2020-05-18 by ser15]

8.1.1.2.2.3-Hly a b gene PCR for Listeria monocytogenes.2013 PCR Protocol SOP  [reviewed by mw16 07072016updated 2020-05-18 by ser15]

8.1.1.2.2.4-inlA ORF and Gene Fragment PCR and Sequencing SOP [reviewed by mw16 07072016]

8.1.1.2.2.5-Modified SigB PCR Protocol-Taq Gold 10uM primers.pdf (MW)

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sigB PCR SOP [updated 2020-01-30 by js2568]

8.1.1.2.2.

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17-

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8.1.1.2.2.14-ribC PCR Protocol for Listeria monocytogenes PCR Protocol  [reviewed by mw16 07072016Degenerate sigB PCR SOP [updated 2020-09-05 by ser15]

8.1.1.2.2.16-PCRs For Quadruple sigma factor Mutant.pdf (Yichang and Veronica)

8.1.1.3-Salmonella PCR

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8.1.1.3

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Salmonella

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PCR

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8.1.1.3.4.2-Salmonella 7 gene MLST Template.xls (check)8.1.1.3.6-InvA colony PCR for Salmonella PCR Protocol SOP [updated 09Aug2016 ajh222020-04-27 amb629]

8.1.1.3.7-PCR identification of common Salmonella serogroups SOP [reviewed by mw16 07072016]

...

8.1.1.4.1-E. coli 6-gene multiplex PCR.pdf

8.1.1.4.24-ipaH colony PCR for Shigella.pdfrpoB PCR Protocol SOP [updated 2020-05-18 by ser15]

8.1.1.4.5-PCR Amplification of Full Length 16s rDNA SOP [updated 18Nov2016 by sjr267]

8.1.1.4.

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5.1-PCR Amplification of 16s rDNA Internal Region SOP [updated 2020-01-30 by js2568]

8.1.1.4.6-ITS PCR for Yeast and Mold SOP [updated 2020-06-26 by rle58]SodA PCR Protocol for S. agalactiae.pdf

8.1.2-Phenotypic Characterization

8.1.3-RFLP

8.1.4-Southern Blot

[archive whole section]

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[see archive

...

]

8.3-DNA preparation, Purification, & Quantification

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8.3.5-Listeria Lysis Methods and Procedures_11_26_13.pdf

8.3.6-PCR product purification using ExoSAP.pdf

8.3.10-Quantification of Purified DNA by NanoDrop.pdf8.3.12-Small-scale phenol-chloroform extraction of chromosomal DNA from Listeria monocytogenes.pdf SOP [updated 2020-11-20 sh988]

8.3.13-Genomic_DNA_Extraction_Qiagen SOP [updated by ajh22 4/28/16]

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8.3.16.4-Example Nextera Library Sample Preparation Spreadsheet Template [reviewed 07Oct2016 by jk2739]

8.3.18-Preparing Cell Lysates for PCR SOP [updated 2020-05-18 by ser15] 

8.4-DNA Sequencing Analysis

Sanger Sequencing

8.4.1-Automated Sequence Editing SOP [updated 2019-10-11 by js2568]

8.4.5-Creating Consensus DNA Sequences using Sequencher SOP [created 2020-04-28 ag67]

8.4.6-rpoB Allelic Type (AT) Assignment SOP [updated 2020-04-28 ag67]

8.4.7-rpoB Database Management SOP [

...

updated 2021-04-01 by ag67]

Whole Genome Sequencing

8.4.2-Quality Control and Assembly SOP [reviewed by jk2739 12/19/2016]

8.4.3-Short Read Submission (SRA) SOP [reviewed by jk2739 12/19/2016]

8.4.4-Assembly submission to NCBI SOP [reviewed by rho2 11/25/2016]

8.5-DNA Recombination & Mutagenesis

...

8.5.20-Whole Mutant-Soeing PCR for L. mono SOP [reviewed by bmb29 4/6/2016]

8.5.21-P22 Transduction of Salmonella Protocol [reviewed by ram524 28Jul2017]

8.5.22-Protocol for Lambda Red Recombinase in Salmonella SOP [updated by ram524 2020-06-01]

8.5.23-Gene Deletion Using I-SceI coupled with Lambda Red Recombination in Salmonella SOP [updated by cq87 2021-07-18]

8.6-Microarray

[see archive below]

8.7-Protein Methods 

8.7.1-Assay for beta-galactosidase activity of B. subtilis transformed with lacZ reporter plasmid Protocol [mw16 checked07072016]

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8.8-RNA Prep & Analysis  

8.8.1 Qiagen RNA Midi Prep with RQ1 DNAse treatment11-RNA Extraction Using TriReagent SOP [updated by arc326 2020-05-18]

8.8.13-Sybr Green qRT-PCR Protocol [updated by arc326 2020-05-18]

8.8.14-RNA Extraction from Salmonella using Tri-Reagent SOP [updated by arc326 2020-05-18 Protocol [reviewed by vg93 Spring 2016]

8.8.515-RNA extraction using Qiagen Midi Kit and Turbo DNAse RNA extraction, clean-up and storage using Turbo DNase for TaqMan.pdf Protocol [reviewed by vg93 Spring 2016]

8.8.10-Taqman- Quantitative Real-Time RT-PCR Protocol Protocol [Updated by vg93 Spring 2016]

8.8.11-RNA Extraction Using TriReagent SOP [reviewed by vg93 4/21/2016]

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extraction from Salmonella using RNeasy Kit SOP [updated 2020-06-01 by ram524]


Training Record

8.1.1.1.5-Guidelines for Primer DesignTrainerJordan Skeens (example)
















































TraineeJordan Skeens (example)
















































Training Date11/02/2020
















































8.1.1.1.5.1-Primer Design ChecklistTrainer

















































Trainee

















































Training Date

















































8.1.1.1.7-Primer Ordering and ReconstitutionTrainer

















































Trainee

















































Training Date

















































8.1.1.1.9-Ten Things That Can Kill Your PCRTrainer

















































Trainee

















































Training Date

















































8.1.1.1.10-Submitting sequences to GenBankTrainer

















































Trainee

















































Training Date

















































8.1.1.1.11-Primer Database Search Protocol.pdfTrainer

















































Trainee

















































Training Date

















































8.1.1.1.17-pGEM Ladder for Size Determination of PCR ProductsTrainer

















































Trainee

















































Training Date

















































8.1.1.1.18-Gel Electrophoresis of PCR ProductTrainerRachel EAlex BSam B














































TraineeZoeSam BMikayla H














































Training Date1/20/21

 

 
















































8.1.1.1.19-PCR Product Purification and Sanger Sequencing SubmissionTrainerRachel EAlex BSam B














































TraineeZoeSam BMikayla H














































Training Date1/21/21

 

 
















































8.1.1.2.2.1-actA PCR Trainer

















































Trainee

















































Training Date

















































8.1.1.2.2.3-Hly a b gene PCR for Listeria monocytogenesTrainer

















































Trainee

















































Training Date

















































8.1.1.2.2.4-inlA ORF and Gene Fragment PCR and SequencingTrainer

















































Trainee

















































Training Date

















































8.1.1.2.2.5-sigB PCRTrainerAlex BSam B















































TraineeSam BMikayla H















































Training Date

 

 

















































8.1.1.2.2.16-PCRs For Quadruple sigma factor Mutant.pdfTrainer

















































Trainee

















































Training Date

















































8.1.1.3.6-InvA colony PCR for SalmonellaTrainer

















































Trainee

















































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8.1.1.3.7-PCR identification of common Salmonella serogroupsTrainer

















































Trainee

















































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8.1.1.3.8-PCR determination of H1 and H2-antigens for SalmonellaTrainer

















































Trainee

















































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8.1.1.4.1-E. coli 6-gene multiplex PCR.pdfTrainer

















































Trainee

















































Training Date

















































8.1.1.4.4-rpoB PCR Protocol TrainerRachel E
















































TraineeZoe
















































Training Date1/20/21
















































8.1.1.4.5-PCR Amplification of Full Length 16s rDNATrainer

















































Trainee

















































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8.1.1.4.5.1-PCR Amplification of 16s rDNA Internal RegionTrainer

















































Trainee

















































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8.1.1.4.6-ITS PCR for Yeast and MoldTrainer

















































Trainee

















































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8.3.10-Quantification of Purified DNA by NanoDrop.pdfTrainer

















































Trainee

















































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8.3.13-Genomic_DNA_ Extraction_QiagenTrainer

















































Trainee

















































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8.3.14-HighThroughput_gDNA_ Extraction_Qiagen _1_28_15.pdfTrainer

















































Trainee

















































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8.3.15-Take3_NucleicAcid_ 96_well_Quantification _01_20_15Trainer

















































Trainee

















































Training Date

















































8.3.16-DNA Preparation for Whole Genome SequencingTrainerSophia H
















































TraineeSam B
















































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8.3.16.1-Step-by-step checklist for Gram+ bacteriaTrainerSophia H
















































TraineeSam B
















































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8.3.16.2-Step-by-step checklist for Gram- bacteriaTrainer

















































Trainee

















































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8.3.16.3-CDC MiSeq Nextera XT Library PreparationTrainer

















































Trainee

















































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8.3.16.4-Example Nextera Library Sample Preparation Spreadsheet Trainer

















































Trainee

















































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8.3.18-Preparing Cell Lysates for PCRTrainerRachel EAlex BSam B














































TraineeZoeSam BMikayla H














































Training Date

(heat lysis) 

1/20/21

 

 
















































8.4.1-Automated Sequence EditingTrainer

















































Trainee

















































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8.4.2-Quality Control and AssemblyTrainer

















































Trainee

















































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8.4.3-Short Read Submission (SRA)Trainer

















































Trainee

















































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8.4.4-Assembly submission to NCBITrainer

















































Trainee

















































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8.4.5-Creating Consensus DNA Sequences using SequencherTrainerAlex BSam B















































TraineeSam BMikayla H















































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8.4.6-rpoB Allelic Type (AT) AssignmentTrainer

















































Trainee

















































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8.5.2-Cloning of DNA FragmentsTrainer

















































Trainee

















































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8.5.9-GUS promoter fusions in Listeria monocytogenesTrainer

















































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8.5.10-Integration of pPL2 derivatives into L. monocytogenes 10403STrainer

















































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8.5.16-pMJG2 vector map.jpgTrainer

















































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8.5.17-pMJG3 vector map.jpgTrainer

















































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8.5.18-pMJG4 vector map.jpgTrainer

















































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8.5.20-Whole Mutant-Soeing PCR for L. monoTrainer

















































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8.5.21-P22 Transduction of SalmonellaTrainer

















































Trainee

















































Training Date

















































8.5.22-Protocol for Lambda Red Recombinase in SalmonellaTrainer

















































Trainee

















































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8.7.1-Assay for beta-galactosidase activity of B. subtilis transformed with lacZ reporter plasmidTrainer

















































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8.7.2-Beta Galactosidase Assay_generalTrainer

















































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8.7.3-His tagged sigma factor protein purification protocolTrainer

















































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8.8.11-RNA Extraction Using TriReagent Trainer

















































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8.8.13-Sybr Green qRT-PCR

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TrainerAlexa Cohn
















































TraineeRory Wang
















































Training Date11/19/21
















































8.8.14_RNAext Salmonella.pdfTrainer

















































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8.1.1.2.2.16