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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.

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

8.1.1.1.5.

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

8.1.1.1.

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7-Primer Ordering and Reconstitution SOP [updated 2020-06-01 ram524]

8.1.1.1.

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

8.1.1.1.

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10-Submitting sequences to GenBank SOP [reviewed by mw16 07072016]

8.1.1.1.

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11-Primer

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Database Search Protocol.pdf

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SOP [updated 2020-06-01 ram524]  

8.1.1.1.

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12-rpoB sigB AT Assignment [MW]

8.1.1.1.

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17-pGEM Ladder for Size Determination of PCR Products SOP [updated by ser15 2020-05-18]

8.1.1.1.

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

8.1.1.1.

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

8.1.1.1.

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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

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]

8.1.1.2-Listeria PCR

8.1.1.2.

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2-

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Listeria PCRs by Gene Name

8.1.1.2.

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2.1-

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actA PCR SOP [updated 2020-05-18 by ser15]

8.1.1.2.

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2.

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3-Hly a b gene PCR for Listeria monocytogenes SOP  [updated 2020-05-18 by ser15]

8.1.1.2.

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2.

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

8.1.1.2.

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2.

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5-

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

8.1.1.2.

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2.

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17-

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Degenerate 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

8.1.1.3.6-

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InvA colony PCR for Salmonella SOP [updated 2020-04-27 amb629]

8.1.1.

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3.7-

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

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...

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8.1.1.4-PCRs for Other Organisms

8.1.1.

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4.1-E.

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coli 6-

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gene multiplex PCR.pdf

8.1.1.4.4-rpoB PCR Protocol SOP [updated 2020-05-18 by ser15]

8.1.1.

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4.

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5-

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

8.1.1.4.

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5.1

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-

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

8.1.1

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.4.6-ITS PCR for Yeast and Mold SOP [updated 2020-06-26 by rle58]

8.1.2-Phenotypic Characterization

8.1.3-RFLP

8.1

...

.4-Southern Blot

[see archive]

8.3-DNA preparation, Purification, & Quantification

8.3.10-Quantification of Purified DNA by NanoDrop SOP [updated 2020-11-20 sh988]

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

8.3.14-HighThroughput_gDNA_Extraction_Qiagen_1_28_15.pdf

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8.3.15-Take3_NucleicAcid_96_well_Quantification_01_20_15  Protocol

[reviewed by mw16 07072016]

8.3.16-DNA Preparation for Whole Genome Sequencing SOP [updated 27Oct2016 by sh988]

8.3.16.1

...

-Step-by-step checklist for Gram+ bacteria Supplemental file [effective 2Sept2016 ajh22]

8.3.16.2-Step-by-step checklist for Gram- bacteria  Supplemental file [effective 13Sept2016 ajh22]

8.3.16.3-CDC MiSeq Nextera XT Library Preparation Reference [reviewed 07Oct2016 by jk2739]

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

8.5.2-Cloning of DNA Fragments Protocol [reviewed 8Aug2016 mw16]

8.5.9-GUS promoter fusions in Listeria monocytogenes Protocol [reviewed 08Aug2016 mw16]

8.5.10-Integration of pPL2 derivatives into L. monocytogenes 10403S Protocol [reviewed 08Aug2016 mw16]

8.5.16-pMJG2 vector map.jpg Plasmid Map [reviewed 08Aug2016 mw16]

8.5.17-pMJG3 vector map.jpg Plasmid Map [reviewed 08Aug2016 mw16]

8.5.18-pMJG4 vector map.jpg Plasmid Map [reviewed 08Aug2016 mw16]

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]

8.7-Protein Methods 

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

8.7.2-Beta Galactosidase Assay_general Protocol [mw16 checked07072016]

8.7.3-His tagged sigma factor protein purification protocol Protocol [mw16 checked07072016]

8.8-RNA Prep & Analysis  

8.8.11-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]

8.8.15-RNA 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

















































Training Date

















































8.1.1.3.7-PCR identification of common Salmonella serogroupsTrainer

















































Trainee

















































Training Date

















































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

















































Trainee

















































Training Date

















































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

















































Training Date

















































8.1.1.4.5.1-PCR Amplification of 16s rDNA Internal RegionTrainer

















































Trainee

















































Training Date

















































8.1.1.4.6-ITS PCR for Yeast and MoldTrainer

















































Trainee

















































Training Date

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

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

8.1.1.2.2.14-RibC PCR Protocol for Listeria monocytogenes [reviewed by mw16 07072016]

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

8.1.1.3-Salmonella PCR

8.1.1.3.4-7 Gene MLST for Salmonella [reviewed by mw16 07072016]

8.1.1.3.4.1-7 gene MLST for Salmonella- Primers and PCR.xls (check)

8.1.1.3.4.2-Salmonella 7 gene MLST Template.xls (check)

8.1.1.3.6-InvA colony PCR for Salm 4-13-10.pdf (Jeff and Jasna - are we using this for Mars project???; update to reflect current protocol if needed)

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

8.1.1.4-PCRs for Other Organisms

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

8.1.1.4.2-ipaH colony PCR for Shigella.pdf

8.1.1.4.3-SodA PCR Protocol for S. agalactiae.pdf

8.1.2-Phenotypic Characterization

8.1.3-RFLP

8.1.4-Southern Blot

[archive whole section]

8.2-DNA Labeling & Modification

[see archive below]

8.3-DNA preparation, Purification, & Quantification

8.3.3-Phenol Chloroform Listeria gDNA Extraction_1_28_2015.pdf

8.3.5-Listeria Lysis Methods and Procedures_11_26_13.pdf

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8.3.10-Quantification of Purified DNA by NanoDrop.pdf

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Trainer

















































Trainee

















































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8.3.13-Genomic_DNA_ Extraction_Qiagen

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Trainer

















































Trainee

















































Training Date

















































8.3.14-HighThroughput_gDNA_ Extraction_Qiagen _1_28_15.pdfTrainer

















































Trainee

















































Training Date

















































8.3.15-Take3_NucleicAcid_ 96_well_Quantification _01_20_15

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Trainer

















































Trainee

















































Training Date

















































8.3.16-DNA Preparation for Whole Genome Sequencing

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TrainerSophia H
















































TraineeSam B
















































Training Date

 


















































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
















































TraineeSam B
















































Training Date

 


















































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

















































Trainee

















































Training Date

















































8.3.16.3-CDC MiSeq Nextera XT Library PreparationTrainer

















































Trainee

















































Training Date

















































8.3.16.4-Example Nextera Library Sample Preparation Spreadsheet Trainer

















































Trainee

















































Training Date

















































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

















































Training Date

8.4-DNA Sequencing Analysis

8.4.1-Automated Sequence Editing SOP [reviewed by sjr267 03/23/2016]

8.5-DNA Recombination & Mutagenesis (MW to review)

8.5.1-Cloning of DNA Flanking Tn917-LTV3.pdf

8.5.2-Cloning of DNA Fragments.pdf

8.5.3-Cloning PCR products for Sequencing using pCR 2.1.pdf

8.5.4-Cloning Protocol for Transformation of E. coli DH5aF.pdf

8.5.5-Electroporation L mono.pdf

8.5.6-Electroporation of E coli.pdf

8.5.7-Electroporation of Mycobacterium paratuberculosis.pdf

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8.5.9-GUS promoter fusions in Listeria monocytogenes

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

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8.5.11-Listeria Genomic Alterations.pdf

8.5.12-Overview-Making a mutant in Listeria monocytogenes.pdf

8.5.13-Part II-Making a mutant in Listeria monocytogenes.pdf

8.5.14-Part III-Making a mutant in Listeria monocytogenes.pdf

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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.jpg

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8.5.20-Whole Mutant-Soeing PCR

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for L. mono

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8.5.21-P22 Transduction of SalmonellaTrainer

















































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8.5.22-Protocol for Lambda Red Recombinase in SalmonellaTrainer

















































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8.6-Microarray

[see archive below]

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

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8.7.2-Beta Galactosidase Assay_general

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8.7.3-His tagged sigma factor protein purification protocol

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8.8-RNA Prep & Analysis  

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8.8.

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11-RNA Extraction Using TriReagent Trainer

















































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8.8.

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13-Sybr Green qRT-PCRTrainerAlexa Cohn
















































TraineeRory Wang
















































Training Date11/19/21
















































8.8.14_RNAext Salmonella.pdfTrainer

















































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8.1.1.2.2.16

...

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

8.8.12-QuantStudio real time PCR protocol [reviewed by vg93]

8.8.13-Sybr Green qRT-PCR [reviewed by vg93]