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=== [[Lab Floor Plan (with list of materials)]] ===
=== [[Detailed Lab Task Descriptions]] ===
== General microbiology protocols ==
== General microbiology protocols ==
===[[Media Recipes]]===
===[[Media Recipes]]===
Line 7: Line 11:
===[[Competition Assays]]===
===[[Competition Assays]]===
===[[Generic PCR]]===
===[[Generic PCR]]===
===[[Gradient PCR]]===
===[[Running DNA Gels]]===
===[[Running DNA Gels]]===
 
===[[Running SDS-PAGE Gels]]===
 
===[[Western Blot]]===
===[[Protein Purification]]===
===[[Protein Sample Concentration]]===
===[[Fixing Cells for Microscope/Flow Cytometry Work]]===


== Cloning and gene manipulation ==
== Cloning and gene manipulation ==
Line 15: Line 23:
===[[Plasmid Purification]]===
===[[Plasmid Purification]]===
===[[Digest and Ligation]]===
===[[Digest and Ligation]]===
===[[Gel Purification]]===
===[[Creating Competent E. coli Cells]]===
===[[Creating Competent E. coli Cells]]===
===[[Transformation]]===
===[[Transformation]]===
===[[Transformation — non-competent E. coli]]===
===[[Gibson Assembly]]===
===[[Gibson Assembly]]===
===[[SOE PCR (Splicing by Overlap-Extension)]]===
===[[Qubit dsDNA Broad Range Assay]]===


== Phyllosphere protocols ==
===[[Preparing Sanger Sequencing (Eurofins)]]===
===[[Preparing Plasmid Sequencing (plasmidsaurus)]]===
 
===[[Creating Lac- E. coli Mutants]]===


===[[Creating Sterile Agar Plates]]===
===[[Sterilization and Germination Protocol for ''Arabidopsis thaliana'' Seeds in Gnotobiotic Experiments]]===
===[[Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments]]===
===[[Growth Stage Phenotype Definitions]]===
===[[Growth Conditions for ''Arabidopsis thaliana'']]===
===[[Measuring Light with HOBO Data Loggers]]===
===[[Inoculation of ''Arabidopsis thaliana'' with Microbes]]===
===[[Removal and DNA Extraction of Phyllosphere Microbes]]===
===[[ARISA]]===
===[[Measuring ''A. thaliana'' Phenotype using FIJI]]===


== ''Streptococcus pneumoniae'' protocols ==
== ''Streptococcus pneumoniae'' protocols ==
===[[Dual Layer Assays]]===
===[[Dual Layer Assays]]===
===[[Streptococcus DNA Extraction]]===
===[[Streptococcus DNA Extraction - Genome Prep]]===
 
===[[Streptococcus CRISPR-Cas9 Editing]]===
===[[Streptococcus Transformation]]===
===[[Streptococcus Growth Curve Protocol]]===
===[[Streptococcus Growth Curve and Cell Count in Liquid Media]]===
===[[Log Phase Growth Curve and Cell Count in Liquid Media]]===
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Original]]===
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Early Producer]]===
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Light and Normal Target Lawns]]===
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria]]===
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria (6-well plate version)]]===
 
== ''Streptococcus mutans'' protocols ==
===[[Streptococcus mutans Growth]]===
===[[Streptococcus mutans Transformation]]===
 
===[[Streptococcus mutans Transformation Media Recipe (SMUR)]]===
 
== ''Myxococcus xanthus'' protocols ==
===[[Media Protocols]]===
===[[Culture Cells from a Frozen Stock]]===
===[[Making a Broth Culture from an Agar Plate]]===
===[[Updated Liquid Biological Waste Disposal Protocol (BSL1)]]===
===[[Generating Frozen Stocks of Strains]]===
===[[Measure Absorbance of M. xanthus Culture]]===
===[[Generate St Curve for OD600 to Cells/mL conversion]]===
===[[Development Assay on Agarose Plates]]===
 
===[[Rehydrating New Primers]]===
 
===[[PCR Amplification from Genome]]===
===[[Ligation of PCR product into TOPO 2.1 vector]]===
===[[Transform competent E. coli cells]]===
===[[Colony PCR to confirm correct insert]]===
===[[Plasmid Isolation with BioBasic Miniprep Kit]]===
===[[EcoRI digest of plasmid]]===
===[[Plate Colonies Using CTTSA]]===
===[[Electroporation and Plating of M. xanthus transformants]]===
===[[M. xanthus genomic DNA extraction with Zymo (yellow) kit]]===
===[[Image Analysis in Fiji]]===
===[[Prepping a Submerged Culture]]===
===[[Heat Fixing and Staining]]===
===[[Propidium Iodide Staining on Agar Plates]]===
===[[Spore Assay]]===
 
== Phage protocols ==
===[[Media and Passaging]]===
===[[Plaque Assays with Soft Agar]]===
===[[Serial Dilutions of Phage]]===
===[[Calculating Virus Titre]]===
===[[Measuring Burst Size]]===
 
== [[Interactions Protocols]] ==
===[[Zone of Inhibition Assay]]===
 
== [[Remote Molecular Biology]] ==
 
 
== [[Effect of Laboratory Protocols on Student Learning]] ==
== Interesting Podcasts to Listen to When Doing Lab Work! ==
 
*This Week in Microbiology
**By Vincent Racaniello
**5 stars! Amazing podcast to learn all about different types of microbiology research! This podcast goes pretty in depth into different current scientific papers so it is a great way to learn about current research.
 
*This Week in Virology
**By Vincent Racaniello
 
*Ologies
**By Alie Ward
**5 stars! Great all around podcast made for a more general audience. There are many different episodes on scientific topics including Environmental Microbiology, Mathematical Biology, and much much more. This is a great podcast to explore different careers and listen to some amazing speakers.
 
*Overheard at National Geographic
**By National Geographic
**4 stars! Great podcast to hear about the many wonders of the world. It also has shorter episodes and is a great way to learn about the world.
 
*Journey to the Micro Cosmos
**3 stars! Short 10 minute episodes about cool microbes.
 
== [[Waste Disposal]] ==
 
== [[Working with GitHub]]==
 


== ''Streptococcus suis'' protocols ==
== ''Streptococcus suis'' protocols ==
Line 47: Line 136:
===[[Measuring Competence : Fixation and Flow Cytometry]]===
===[[Measuring Competence : Fixation and Flow Cytometry]]===


== [[Remote Molecular Biology]] ==
== ''Arabidopsis thaliana'' protocols ==


===[[Creating Sterile Agar Plates]]===
===[[Sterile Seeding Protocol]]===
===[[Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments]]===
===[[Growth Stage Phenotype Definitions]]===
===[[Growth Conditions for ''Arabidopsis thaliana'']]===
===[[Measuring Light with HOBO Data Loggers]]===
===[[Inoculation of ''Arabidopsis thaliana'' with Microbes]]===
===[[Removal and DNA Extraction of Phyllosphere Microbes]]===
===[[ARISA]]===
===[[Measuring ''A. thaliana'' Phenotype using FIJI by Hand]]===
===[[DNeasy PowerSoil Protocol]]===
===[[Fiji Measurement]]===
===[[Making Boxes]]===
===[[Growing ''A. thaliana'' for Seed Harvest]]===
===[[Growing ''A. thaliana'' in Cut Pipet Tips]]===


== [[Effect of Laboratory Protocols on Student Learning]] ==


== Cambridge protocols ==
== Cambridge protocols ==
=== [[Storage buffer]] ===
=== [[Storage buffer]] ===
=== [[transformation]] ===
=== [[transformation of R5(2)-mCh-FL-BST and ]] ===
 
=== Transformation of R5(2)-mCh-FL-BST and R5(2)-mCh-H10-BST ===
 
==== protocol: ====
#Thaw BL21(DE3) Cells on ice
#Pipette 50 μL of cells into transformation tube
#Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
##170 ng/μl for R52-mCh-FL-BST
##Unknown concentration for R52-mCh-H10-BST
#Place mixture on ice for 30 mins.
##Begin to warm up LB Broth
#Heat shock at exactly 42°C for 10 secs.
##Used water bath
##Actual time: ~15 seconds
#Place on ice for 5 mins.
#Pipette 950 μL of room temperature LB Broth into mixture.
#Incubate at 37°C with 250 rpm for 60 mins.
##Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
#Mix cells thoroughly and spread 100 μL of the mixture onto a plate
##One plate for either plasmid
##Leftover transformation mixtures stored in 4°C fridge
#Incubate overnight at 37°C
 
Results:
The selection plates showed no colonies.
 
Troubleshoot:
Increase heat shock time from 10 seconds to 45 seconds
Additional transformation with pUC19
Plate the transformed cells with non-selection Agar plates along side selection plates
Use heat block instead of water bath
 
Second Attempt - Protocol:
#Thaw BL21(DE3) Cells on ice
#Pipette 50 μL of cells into transformation tube
##Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
##170 ng/μl for R52-mCh-FL-BST
##Unknown concentration for R52-mCh-H10-BST
163 pg/μl for pUC19
Place mixture on ice for 30 mins.
Begin to warm up LB Broth
Heat shock at exactly 42°C for 45 secs.
Used heat block instead of water bath
Place on ice for 5 mins.
Pipette 950 μL of room temperature LB Broth into mixture.
Incubate at 37°C with 225 rpm for 60 mins.
Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
Mix cells thoroughly and spread 100 μL of the mixture onto a plate
One Agar plate w/ KAN for BST plasmids
One Agar plate w/ AMP for pUC19
One Agar plate w/o KAN for all 3 plasmids
Leftover transformation mixtures stored in 4°C fridge
Incubate overnight at 37°C
 
=== [[expression]] ===
=== [[expression]] ===
=== [[lysis and immobilization]] ===
=== [[lysis and immobilization]] ===


==[[Bio320 Microbe Species Wikipedia Pages]]==


== Getting started with MediaWiki ==
== Getting started with MediaWiki ==

Latest revision as of 11:06, 19 June 2024

Lab Floor Plan (with list of materials)

Detailed Lab Task Descriptions

General microbiology protocols

Media Recipes

Reagent Recipes

Working with Antibiotics

Freezing -80 Stocks

Freezing Aliquots

Competition Assays

Generic PCR

Gradient PCR

Running DNA Gels

Running SDS-PAGE Gels

Western Blot

Protein Purification

Protein Sample Concentration

Fixing Cells for Microscope/Flow Cytometry Work

Cloning and gene manipulation

Commonly Used Plasmids

Plasmid Purification

Digest and Ligation

Gel Purification

Creating Competent E. coli Cells

Transformation

Transformation — non-competent E. coli

Gibson Assembly

SOE PCR (Splicing by Overlap-Extension)

Qubit dsDNA Broad Range Assay

Preparing Sanger Sequencing (Eurofins)

Preparing Plasmid Sequencing (plasmidsaurus)

Creating Lac- E. coli Mutants

Streptococcus pneumoniae protocols

Dual Layer Assays

Streptococcus DNA Extraction - Genome Prep

Streptococcus CRISPR-Cas9 Editing

Streptococcus Transformation

Streptococcus Growth Curve Protocol

Streptococcus Growth Curve and Cell Count in Liquid Media

Log Phase Growth Curve and Cell Count in Liquid Media

Streptococcus Bacteriocin (Dual Layer) Assays - Original

Streptococcus Bacteriocin (Dual Layer) Assays - Early Producer

Streptococcus Bacteriocin (Dual Layer) Assays - Light and Normal Target Lawns

Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria

Streptococcus Bacteriocin (Dual Layer) Assays - Finding Producer-Resistant Target Bacteria (6-well plate version)

Streptococcus mutans protocols

Streptococcus mutans Growth

Streptococcus mutans Transformation

Streptococcus mutans Transformation Media Recipe (SMUR)

Myxococcus xanthus protocols

Media Protocols

Culture Cells from a Frozen Stock

Making a Broth Culture from an Agar Plate

Updated Liquid Biological Waste Disposal Protocol (BSL1)

Generating Frozen Stocks of Strains

Measure Absorbance of M. xanthus Culture

Generate St Curve for OD600 to Cells/mL conversion

Development Assay on Agarose Plates

Rehydrating New Primers

PCR Amplification from Genome

Ligation of PCR product into TOPO 2.1 vector

Transform competent E. coli cells

Colony PCR to confirm correct insert

Plasmid Isolation with BioBasic Miniprep Kit

EcoRI digest of plasmid

Plate Colonies Using CTTSA

Electroporation and Plating of M. xanthus transformants

M. xanthus genomic DNA extraction with Zymo (yellow) kit

Image Analysis in Fiji

Prepping a Submerged Culture

Heat Fixing and Staining

Propidium Iodide Staining on Agar Plates

Spore Assay

Phage protocols

Media and Passaging

Plaque Assays with Soft Agar

Serial Dilutions of Phage

Calculating Virus Titre

Measuring Burst Size

Interactions Protocols

Zone of Inhibition Assay

Remote Molecular Biology

Effect of Laboratory Protocols on Student Learning

Interesting Podcasts to Listen to When Doing Lab Work!

  • This Week in Microbiology
    • By Vincent Racaniello
    • 5 stars! Amazing podcast to learn all about different types of microbiology research! This podcast goes pretty in depth into different current scientific papers so it is a great way to learn about current research.
  • This Week in Virology
    • By Vincent Racaniello
  • Ologies
    • By Alie Ward
    • 5 stars! Great all around podcast made for a more general audience. There are many different episodes on scientific topics including Environmental Microbiology, Mathematical Biology, and much much more. This is a great podcast to explore different careers and listen to some amazing speakers.
  • Overheard at National Geographic
    • By National Geographic
    • 4 stars! Great podcast to hear about the many wonders of the world. It also has shorter episodes and is a great way to learn about the world.
  • Journey to the Micro Cosmos
    • 3 stars! Short 10 minute episodes about cool microbes.

Waste Disposal

Working with GitHub

Streptococcus suis protocols

Streptococcus suis Transformation

Measuring Absorbance in Streptococcus

Streptococcus DNA Extraction

Streptococcus Competence Induction

Peptide Synthesis

Peptide Cleavage

Mass Spectrometery

Plate Reader Assay and Growth Curve

Measuring Competence : Fixation and Flow Cytometry

Arabidopsis thaliana protocols

Creating Sterile Agar Plates

Sterile Seeding Protocol

Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments

Growth Stage Phenotype Definitions

Growth Conditions for ''Arabidopsis thaliana''

Measuring Light with HOBO Data Loggers

Inoculation of ''Arabidopsis thaliana'' with Microbes

Removal and DNA Extraction of Phyllosphere Microbes

ARISA

Measuring ''A. thaliana'' Phenotype using FIJI by Hand

DNeasy PowerSoil Protocol

Fiji Measurement

Making Boxes

Growing ''A. thaliana'' for Seed Harvest

Growing ''A. thaliana'' in Cut Pipet Tips

Cambridge protocols

Storage buffer

transformation of R5(2)-mCh-FL-BST and

expression

lysis and immobilization

Bio320 Microbe Species Wikipedia Pages

Getting started with MediaWiki

Consult the User's Guide for information on using the wiki software.

Retrieved from "http://microbes.sites.haverford.edu/MEE_wiki/index.php?title=Main_Page&oldid=3424"