8-Molecular Biology Protocols

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]

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

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

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

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

8.1.1.1.11-Primer Database Search Protocol.pdf SOP [updated 2020-06-01 ram524]  

8.1.1.1.12-rpoB sigB AT Assignment [MW]

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

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

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

8.1.1.1.20-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

8.1.1.2.2-Listeria PCRs by Gene Name

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

8.1.1.2.2.3-Hly a b gene PCR for Listeria monocytogenes SOP  [updated 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-sigB PCR SOP [updated 2020-01-30 by js2568]

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

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

8.1.1.3.8-PCR determination of H1 and H2-antigens for Salmonella SOP [reviewed by mw16 07072016]

8.1.1.4-PCRs for Other Organisms

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

8.1.1.4.4-rpoB 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]

8.1.1.4.5.1-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]

8.1.2-Phenotypic Characterization

[see archive]

8.1.3-RFLP

[see archive]

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

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]

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 [created 2020-06-01 by ram524]

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