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8.1- Characterization & DNA amplification

8.1.1-PCR
8.1.1.1-General PCR Methods & Guidelines

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

8.1

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.1.1.5.1-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-

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Primer

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Database Search Protocol.pdf 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]

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.

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2.

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

8.1.1

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.3-Salmonella PCR

8.1.1.

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

8.1.1

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

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8.1.1.

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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

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pdf

8.1.1.

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4.

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4-

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rpoB PCR

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Protocol SOP [updated 2020-05-18 by ser15]

8.1.1.

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4.

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

8.1.1.

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4.

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5.1-

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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.

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6-

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ITS PCR

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for Yeast and Mold SOP [updated 2020-06-26 by rle58]

8.1.2-Phenotypic Characterization

8.1.3-RFLP

8.1.

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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

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

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8.1.1.2.2.4-inlA ORF and Gene Fragment PCR and Sequencing.pdf

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

8.1.1.2.2.6-Multiplex PCR for inlH screening.pdf

8.1.1.2.2.7-Nested hly PCR for Listeria monocytogenes.pdf

8.1.1.2.2.8-Protocol for sequencing actA gene most polymorphic region.pdf

8.1.1.2.2.9-Protocol for Sequencing actA gene region with best discrimination.pdf

8.1.1.2.2.10-Protocol for Sequencing inlA gene region with the best discrimination.pdf

8.1.1.2.2.11-Prs PCR Protocol for Listeria monocytogenes.pdf

8.1.1.2.2.12-PurM PCR Protocol for lineage II Listeria monocytogenes.pdf

8.1.1.2.2.13-PurM PCR Protocol for Listeria monocytogenes.pdf

8.1.1.2.2.14-RibC PCR Protocol for Listeria monocytogenes.pdf

8.1.1.2.2.15-SigB PCR Protocol GoTaq.pdf

8.1.1.3-Salmonella PCR

8.1.1.3.1-fimA PCR and DNA sequencing of Salmonella enterica for 3gene MLST.pdf

8.1.1.3.2-manB PCR and DNA sequencing of Salmonella enterica for 3gene MLST.pdf

8.1.1.3.3-mdh PCR and DNA sequencing of Salmonella enterica for 3gene MLST.pdf

8.1.1.3.4-7 Gene MLST for Salmonella.pdf

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

8.1.1.3.4.2-Salmonella 7 gene MLST Template.xls

8.1.1.3.5-PCR detection of the presence or absence of antibiotic resistance genes in Salmonella.pdf

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

















































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8.1.1.1.7-Primer Ordering and ReconstitutionTrainer

















































Trainee

















































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8.1.1.1.9-Ten Things That Can Kill Your PCRTrainer

















































Trainee

















































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8.1.1.1.10-Submitting sequences to GenBankTrainer

















































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8.1.1.1.11-Primer Database Search Protocol.pdfTrainer

















































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8.1.1.1.17-pGEM Ladder for Size Determination of PCR ProductsTrainer

















































Trainee

















































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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

















































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8.1.1.2.2.3-Hly a b gene PCR for Listeria monocytogenesTrainer

















































Trainee

















































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8.1.1.2.2.4-inlA ORF and Gene Fragment PCR and SequencingTrainer

















































Trainee

















































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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

















































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8.1.1.3.6-InvA colony PCR for

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SalmonellaTrainer

















































Trainee

















































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8.1.1.3.7-PCR identification of common Salmonella serogroups

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Trainer

















































Trainee

















































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

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Trainer

















































Trainee

















































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

















































Trainee

















































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8.1.1.4.

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4-

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rpoB PCR Protocol TrainerRachel E
















































TraineeZoe
















































Training Date1/20/21
















































8.1.1.4.

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5-

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PCR Amplification of Full Length 16s rDNATrainer

















































Trainee

















































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8.1.2-Phenotypic Characterization

8.1.2.1-Semi-Quantitative Hemolysis Assay for Listeria.pdf

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8.1

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.1

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4

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.5.1-PCR Amplification of 16s rDNA Internal RegionTrainer

















































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8.1

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1.4.

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8.2-DNA Labeling & Modification

8.2.1-3' end labeling of oligonucleotides.pdf

8.2.2-Kinasing and DIG labeling of LCR primer.pdf

8.2.3-Poly-T tailing of oligonucleotides.pdf

8.2.4-Protocol for Fluorescein labeling of oligonucleotides carrying an aminolink.pdf

8.2.5-Protocol for Geneclean (USB).pdf

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6-ITS PCR for Yeast and MoldTrainer

















































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8.3.10-Quantification of Purified DNA by NanoDrop.pdfTrainer

















































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8.3.

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13-

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Genomic_DNA_ Extraction_QiagenTrainer

















































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8.3.

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14-HighThroughput_gDNA_ Extraction_Qiagen _1_28_15.pdfTrainer

















































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8.3.

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15-Take3_NucleicAcid_ 96_well_Quantification _01_20_15Trainer

















































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8.3.

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16-DNA Preparation

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for Whole Genome SequencingTrainerSophia H
















































TraineeSam B
















































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8.3

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.16.1-Step-by-step checklist for Gram+ bacteriaTrainerSophia H
















































TraineeSam B
















































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

















































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8.3.

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16.3

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CDC MiSeq Nextera XT Library PreparationTrainer

















































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8.3.

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16.4-Example Nextera Library Sample Preparation Spreadsheet Trainer

















































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8.3.

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18-Preparing Cell Lysates for PCRTrainerRachel EAlex BSam B














































TraineeZoeSam BMikayla H














































Training Date

(heat lysis) 

1/20/21

 

 

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8.3.11-Single Stranded DNA preparation from M13.pdf

8.3.12-Small-scale phenolchloroform extraction of chromosomal DNA from Listeria monocytogenes.pdf

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8.4.1-

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Automated Sequence EditingTrainer

















































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8.4.2-

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Quality Control and AssemblyTrainer

















































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8.4.3-

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Short Read Submission (SRA)Trainer

















































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8.4.4-

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Assembly submission to NCBITrainer

















































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8.4.5-

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Creating Consensus DNA Sequences using SequencherTrainerAlex BSam B















































TraineeSam BMikayla H















































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8.4.6-

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rpoB Allelic Type (AT) AssignmentTrainer

















































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8.

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5.

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2-

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Cloning

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of DNA FragmentsTrainer

















































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5.9-GUS promoter fusions in Listeria monocytogenes

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

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8.5.

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8.4.11-Listeria Genomic Alterations.pdf

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

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

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

8.4.15-Part IV-Making a mutant in Listeria monocytogenes.pdf

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16-pMJG2 vector map.jpgTrainer

















































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5.17-pMJG3 vector map.jpgTrainer

















































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5.18-pMJG4 vector map.jpg

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8.

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5.20-Whole Mutant-

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Soeing PCR for L. mono

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8.5.

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21-P22 Transduction of SalmonellaTrainer

















































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8.5.

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22-Protocol for Lambda Red Recombinase in SalmonellaTrainer

















































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8.7

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8.5.4-Microarray Slide Blocking.pdf

8.6-Protein Methods

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

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7.2-Beta Galactosidase Assay

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_generalTrainer

















































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7.3-His tagged sigma factor protein purification protocol

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8.

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11-RNA Extraction Using TriReagent Trainer

















































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8.8.13-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

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8.7.2-Isolation of RNA from Listeria monocytogenes infected mammalian cells.pdf

8.7.3-Preparation of infected tissue culture cells for bacterial RNA collection.pdf

8.7.4-Preperation of Listeria RNA.pdf

8.7.5-RNA extraction, clean-up and storage using Turbo DNase for TaqMan.pdf

8.7.6-RNA Prep with RNeasy Midi Kit instructions.pdf

8.7.7-RNeasy kit total RNA isolation from Listeria monocytogenes with on-column DNase treatment.pdf

8.7.8-RT-PCR for the detection of cytokine mRNA.pdf

8.7.9-Synthesis of large amounts of RNA.pdf

8.7.10-Taqman- Quantitative Real-Time RT-PCR Protocol.pdf