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=== [[Lab Floor Plan (with list of materials)]] ===
=== [[Lab Floor Plan (with list of materials)]] ===
=== [[Detailed Lab Task Descriptions]] ===


== General microbiology protocols ==
== General microbiology protocols ==
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===[[Gradient PCR]]===
===[[Gradient PCR]]===
===[[Running DNA Gels]]===
===[[Running DNA Gels]]===
===[[Running SDS-PAGE Gels]]===
===[[Western Blot]]===
===[[Protein Purification]]===
===[[Protein Purification]]===
===[[Protein Sample Concentration]]===
===[[Protein Sample Concentration]]===
===[[Fixing Cells for Microscope/Flow Cytometry Work]]===


== Cloning and gene manipulation ==
== Cloning and gene manipulation ==
Line 25: Line 29:
===[[Transformation — non-competent E. coli]]===
===[[Transformation — non-competent E. coli]]===
===[[Gibson Assembly]]===
===[[Gibson Assembly]]===
===[[SOE PCR (Splicing by Overlap-Extension)]]===
===[[Qubit dsDNA Broad Range Assay]]===
===[[Qubit dsDNA Broad Range Assay]]===


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===[[Creating Lac- E. coli Mutants]]===
===[[Creating Lac- E. coli Mutants]]===


== ''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]]===


== ''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 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)]]===


== Reagents Needed==
== ''Streptococcus mutans'' protocols ==
* pSD05 plasmid (P-80 in our freezer)
===[[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]]===


== Designing gRNA Cut Site==
== [[Remote Molecular Biology]] ==
# Use Benchling CRISPR guide along with the specific genome that you want to change. Find the gene what we want to disrupt, and have the CRISPR guide xxxxx
# Design 8 primers:
## Two primers that, when put together, will create the proper gRNA guide RNA. There's not PCR needed for this; it is instead annealing the two primers together. Primers should be in the form XXXXXX and XXXXXX
## Two PCR primers that will amplify 500bp-1.5kb of the region before the cut site, which will act as a homologous recombination site. It does not need to be directly next to the cute site (and probably should not be). So, this should be upstream of gene A if we are trying to get rid of gene A.
## Two PCR primers that will amplify 500bp-1.5kb of the region after the cut site, which will act as a homologous recombination site. So, this should be downstream of gene A if we are trying to get rid of gene A.
## Two primers that will amplify the region across the cut site; ideally, we want a small PCR product if the procedure works, and a longer PCR product if the strain remains untransformed.
## Check with Eric about these primers and have him order them.




== [[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.


* CTM (Complete Transformation Medium) pH 6.8
*This Week in Virology
* CTM pH 7.8
**By Vincent Racaniello
* CSP-1 peptide (in the -80 freezer; 20ul aliquots of 100uM) or CSP-2 peptide (which we do not have). Talk to Eric if you are not working with a D39 derivative.


== Complete Transformation Medium ==
*Ologies
* 3g Tryptic Soy Broth
**By Alie Ward
* 0.1g yeast extract
**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.
* Fill up to 100ml MilliQ water and autoclave
* Add to a final concentration filter sterilized 1mM CaCl2 (found on chemical shelf), filter sterilized 0.2% BSA (Bovine Serum Albumin), and filter sterilized 1X trace mineral solution (found on chemical shelf)


== Competence Protocol ==
*Overheard at National Geographic
# Freshly grow up single colonies on a blood TSA plate of the strain to be transformed.
**By National Geographic
# Select one colony and grow in 3ml CTM pH 6.8 until OD 0.3, which is 0.39 Absorbance
**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.  
# Preheat a microcentrifuge tube of 270ul CTM pH 7.8 to 37 degrees using the hot block.
# Add CSP-1 peptide to this tube to at least 100 ng/ml eventual final concentration. We use 2ul of the CSP-1 aliquot, which brings the concentration to 228ng/ml.
# Add DNA to 1 ug/ml final concentration — so 300ng. If this is too much DNA, it might work with half of the amount — 150ng of DNA.
# Add 30ul of grown cells (a 1:10 dilution).
# Vortex
# Incubate at 37 degrees using the hot block for 60 minutes.
# Plate cells on a blood TSA plate that has appropriate antibiotics in it. Use sterile cotton swab to spread the cell mixture.
# Incubate the plate overnight in the 37 degrees C 5% CO2 incubator.


== Competence Protocol with CRISPR ==
*Journey to the Micro Cosmos
* As above, but add the editing construct at a final concentration of 0.7 - 2.5 ug/ml (210ng - 750ng total DNA)
**3 stars! Short 10 minute episodes about cool microbes.
* Incubate at 37 degrees using the hot block for 20 minutes.
 
* Add the CRISPR targeting construct at a final concentration of 0.7 - 2.5 ug/ml (mirroring the first set of DNA), and vortex.
== [[Waste Disposal]] ==
* Incubate at 37 degrees using the hot block for 40 minutes.
 
== [[Working with GitHub]]==


===[[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 suis'' protocols ==
== ''Streptococcus suis'' protocols ==
Line 121: Line 136:
===[[Measuring Competence : Fixation and Flow Cytometry]]===
===[[Measuring Competence : Fixation and Flow Cytometry]]===


== ''Streptococcus mutans'' protocols ==
== ''Arabidopsis thaliana'' protocols ==
===[[Streptococcus mutans Growth]]===
===[[Streptococcus mutans Transformation]]===


== [[Interactions Protocols]] ==
===[[Creating Sterile Agar Plates]]===
===[[Zone of Inhibition Assay]]===
===[[Sterile Seeding Protocol]]===
 
===[[Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments]]===
== [[Remote Molecular Biology]] ==
===[[Growth Stage Phenotype Definitions]]===
 
===[[Growth Conditions for ''Arabidopsis thaliana'']]===
 
===[[Measuring Light with HOBO Data Loggers]]===
== [[Effect of Laboratory Protocols on Student Learning]] ==
===[[Inoculation of ''Arabidopsis thaliana'' with Microbes]]===
== Interesting Podcats to Listen to When Doing Lab Work! ==
===[[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]]===


== [[Waste Disposal]] ==


== Cambridge protocols ==
== Cambridge protocols ==
Line 141: Line 160:
=== [[expression]] ===
=== [[expression]] ===
=== [[lysis and immobilization]] ===
=== [[lysis and immobilization]] ===
== Detailed Lab Task Descriptions ==
=== [[Serological Pipetes]] ===
=== [[Media]] ===
=== [[Medical Waste]] ===
=== [[Tips/Tubes]] ===
=== [[Glassware (no 50 mL flasks)]] ===
=== [[Just Flasks]] ===
=== [[Water/Ethanol]] ===
=== [[Balance/Microwave/Gel Box/Virkon]] ===


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

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"