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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 ==
Line 11: Line 13:
===[[Gradient PCR]]===
===[[Gradient PCR]]===
===[[Running DNA Gels]]===
===[[Running DNA Gels]]===
===[[Running SDS-PAGE Gels]]===
===[[Western Blot]]===
===[[Protein Purification]]===
===[[Protein Purification]]===
===[[Protein Sample Concentration]]===
===[[Fixing Cells for Microscope/Flow Cytometry Work]]===


Procedure:
== Cloning and gene manipulation ==
===[[Commonly Used Plasmids]]===
===[[Plasmid Purification]]===
===[[Digest and Ligation]]===
===[[Gel Purification]]===


LOCATION: MEE LAB
===[[Creating Competent E. coli Cells]]===
Kept at 4ºC — In the MEE fridge
===[[Transformation]]===
Put 30-40ml aliquot of lysis buffer in 50 mL tube on ice in 100 mL beaker
===[[Transformation — non-competent E. coli]]===
Add ? mL of lysis buffer to each cell pellet
===[[Gibson Assembly]]===
Turn on large centrifuge and set it to 4ºC (do this now so it can reach this temp when needed)
===[[SOE PCR (Splicing by Overlap-Extension)]]===
Resuspend via vortex until smooth to avoid clumps of pellet. Very important!
===[[Qubit dsDNA Broad Range Assay]]===
Go down Bio superlab on first cloor of KINSC


===[[Preparing Sanger Sequencing (Eurofins)]]===
===[[Preparing Plasmid Sequencing (plasmidsaurus)]]===


CHANGE LOCATION: Bio Superlab
===[[Creating Lac- E. coli Mutants]]===
Put on ear protection!
Put up sign on freezer door that indicates sonicator is turned on. Important Saftey Measure
Clean sonicator probe using ethanol and dry with Kimwipe before use.
Fully immerse sonicator tip in beaker with cell, but make sure the tip does not touch any beaker walls.
Sonicator operating settings:
Put sonitcater at power level of 40%
Operate: 30 seconds on, 30 seconds off. Do for 10 minutes total ( 5 min sonication overall)
Spin down in **chilled** ultra-centrifuge
Use smaller tubes (these will be located under the swinging bucket centrifuge - specify spatial location later)
14000 RPM for 45 minutes
While spinning, start next steps


Column Equilibration


CHANGE LOCATION: MEE LAB
== ''Streptococcus pneumoniae'' protocols ==
Get Nickel agarose high density beads from 4ºC MEE fridge — shake well!
===[[Dual Layer Assays]]===
To equilibrate the with lysis buffer:
===[[Streptococcus DNA Extraction - Genome Prep]]===
Get 15 mL conical and add about 10 mL of lysis buffer (make sure its chiled)
Need 4 mL of slurry total. (slurry is the term for a combianation of resin beads and ethanol)
1 mL of resin beads == 15 mg protein (good for our 2 L)
Resin is in 50% ethanol, so need 4 mL of resin
NOTE: Do not leave out of refrigerator
Use spinning bucket centrifuge, 3000 RPM for 2 minutes (be sure to balance properly)  - what’s the quick balance method?
Discard ethanol
Add 2 mL lysis buffer
Mix and spin down
Carry out Steps d-f a total of 4 times total
Remove lysis buffer so that only the resin beads remain (this is to make sure all ethanol is removed)


Gentilly pour cell lysate from Step 12 into a 50 mL tube. Leave 1 mL so that cell debris does not come out (very important step)
===[[Streptococcus CRISPR-Cas9 Editing]]===
Add resin beads to the 50 mL tube and use cell lysate to resuspend the resin beads (in its 15 mL tube?)  
===[[Streptococcus Transformation]]===
Use cell lysate form 15 mL tubes to resuspend the resin beads 
===[[Streptococcus Growth Curve Protocol]]===
Pipette up and down
===[[Streptococcus Growth Curve and Cell Count in Liquid Media]]===
mix all resin into the lysate.
===[[Log Phase Growth Curve and Cell Count in Liquid Media]]===
Wash walls with lysate to get all resin out.
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Original]]===
Seal with parafilm.
===[[Streptococcus Bacteriocin (Dual Layer) Assays - Early Producer]]===
Place flat on orbital shaker for 30 minutes (Good stopping point for lunch)
===[[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)]]===


Disposable Column
== ''Streptococcus mutans'' protocols ==
Get the disposable column and snap off the end. Get the lid and stopper ready.
===[[Streptococcus mutans Growth]]===
===[[Streptococcus mutans Transformation]]===


CHANGE LOCATION: Cold Room in Bio Superlab
===[[Streptococcus mutans Transformation Media Recipe (SMUR)]]===
Work in the cold room in Bio super lab; set up column with clamps holding it in place (example picture)
Pour in resin and proteins. Rotate tube to get resin out
NOTE: Avoid forming air bubbles
Open bottom and let all liquid pour through.
Take small volume (2 uL) of flow-through in the Eppendorf tube and measure absorbance.
This volume is labelled FLOUGH THOUGH Start
Take FLOUGH THOUGH Start to Nanodrop to obtain absorbance readings
Th aim is get the column to have strong 260 absorbance due to oligonucleotides:
Keep adding lysis buffer until 260 absorbance is zero. This will take about  9-10 volumes
NOTE: when adding buffer, use a Pasteur pipette to apply it to the walls of the column so as not to distub the resin
Don’t let the resin get dry, keep it hydrated at all times


Measuring Absorbance for Washes
== ''Myxococcus xanthus'' protocols ==
Add 200 mL (?) wash buffer
===[[Media Protocols]]===
Obtain absorbance readings from these washes in the same way that was done for the flowthrough in Steps e-f
===[[Culture Cells from a Frozen Stock]]===
Th aim is get the column to have strong 260 absorbance due to oligonucleotides:
===[[Making a Broth Culture from an Agar Plate]]===
Keep adding lysis buffer until 260 absorbance is zero. This will take about  9-10 volumes
===[[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]]===


Nanodrop  2 uL "protein" at 280nm,
===[[PCR Amplification from Genome]]===
"Overlay spectra" between readouts.  
===[[Ligation of PCR product into TOPO 2.1 vector]]===
Use lysis buffer for blank. Measure blank to makes sure there Nanodrop is clean and that there’s no preexisting absorbance.
===[[Transform competent E. coli cells]]===
Note: Looking for strong 260 peak (~0.85)
===[[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]]===


==Goal==
== Phage protocols ==
To prepare plasmids from a strain of bacteria
===[[Media and Passaging]]===
===[[Plaque Assays with Soft Agar]]===
===[[Serial Dilutions of Phage]]===
===[[Calculating Virus Titre]]===
===[[Measuring Burst Size]]===


== [[Interactions Protocols]] ==
===[[Zone of Inhibition Assay]]===


==Growing Up Strains==  
== [[Remote Molecular Biology]] ==
# From glycerol stocks in -20°C freezer, get the strains you want to work with.
#* D39
#* Hermans 930
#* Pmen 4
# Day 1:
#* Get Blood Plates (1 per strain), inoculation loops, tube holder. Bring everything into the hood.
#* Using an inoculation loop, streak for single colonies onto the entire plate. Obviously change loops between




a) seems like it is roughly 10 ul, your master mix is probably fine.
== [[Effect of Laboratory Protocols on Student Learning]] ==
b) falls a little short of 10 ul, maybe the volume distribution in the 8 tubes was not equal. You can probably proceed.
== Interesting Podcasts to Listen to When Doing Lab Work! ==
c) is close to 0 ul, you might have not added the proper volume for all reagents.
  i) You might have to redo the master mix to avoid an
  unsuccessful PCR
 
Procedure:
 
LOCATION: MEE LAB
Kept at 4ºC — In the MEE fridge
Put 30-40ml aliquot of lysis buffer in 50 mL tube on ice in 100 mL beaker
Add ? mL of lysis buffer to each cell pellet
Turn on large centrifuge and set it to 4ºC (do this now so it can reach this temp when needed)
Resuspend via vortex until smooth to avoid clumps of pellet. Very important!
Go down Bio superlab on first cloor of KINSC
 
 
CHANGE LOCATION: Bio Superlab
Put on ear protection!
Put up sign on freezer door that indicates sonicator is turned on. Important Saftey Measure
Clean sonicator probe using ethanol and dry with Kimwipe before use.
Fully immerse sonicator tip in beaker with cell, but make sure the tip does not touch any beaker walls.
Sonicator operating settings:
Put sonitcater at power level of 40%
Operate: 30 seconds on, 30 seconds off. Do for 10 minutes total ( 5 min sonication overall)
Spin down in **chilled** ultra-centrifuge
Use smaller tubes (these will be located under the swinging bucket centrifuge - specify spatial location later)
14000 RPM for 45 minutes
While spinning, start next steps
 
Column Equilibration


CHANGE LOCATION: MEE LAB
*This Week in Microbiology
Get Nickel agarose high density beads from 4ºC MEE fridge — shake well!
**By Vincent Racaniello
To equilibrate the with lysis buffer:
**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.
Get 15 mL conical and add about 10 mL of lysis buffer (make sure its chiled)
Need 4 mL of slurry total. (slurry is the term for a combianation of resin beads and ethanol)
1 mL of resin beads == 15 mg protein (good for our 2 L)
Resin is in 50% ethanol, so need 4 mL of resin
NOTE: Do not leave out of refrigerator
Use spinning bucket centrifuge, 3000 RPM for 2 minutes (be sure to balance properly)  - what’s the quick balance method?
Discard ethanol
Add 2 mL lysis buffer
Mix and spin down
Carry out Steps d-f a total of 4 times total
Remove lysis buffer so that only the resin beads remain (this is to make sure all ethanol is removed)


Gentilly pour cell lysate from Step 12 into a 50 mL tube. Leave 1 mL so that cell debris does not come out (very important step)
*This Week in Virology
Add resin beads to the 50 mL tube and use cell lysate to resuspend the resin beads (in its 15 mL tube?)
**By Vincent Racaniello
Use cell lysate form 15 mL tubes to resuspend the resin beads 
Pipette up and down
mix all resin into the lysate.
Wash walls with lysate to get all resin out.
Seal with parafilm.
Place flat on orbital shaker for 30 minutes (Good stopping point for lunch)


Disposable Column
*Ologies
Get the disposable column and snap off the end. Get the lid and stopper ready.
**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.  


CHANGE LOCATION: Cold Room in Bio Superlab
*Overheard at National Geographic
Work in the cold room in Bio super lab; set up column with clamps holding it in place (example picture)
**By National Geographic
Pour in resin and proteins. Rotate tube to get resin out
**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.  
NOTE: Avoid forming air bubbles
Open bottom and let all liquid pour through.
Take small volume (2 uL) of flow-through in the Eppendorf tube and measure absorbance.
This volume is labelled FLOUGH THOUGH Start
Take FLOUGH THOUGH Start to Nanodrop to obtain absorbance readings
Th aim is get the column to have strong 260 absorbance due to oligonucleotides:
Keep adding lysis buffer until 260 absorbance is zero. This will take about  9-10 volumes
NOTE: when adding buffer, use a Pasteur pipette to apply it to the walls of the column so as not to distub the resin
Don’t let the resin get dry, keep it hydrated at all times


Measuring Absorbance for Washes
*Journey to the Micro Cosmos
Add 200 mL (?) wash buffer
**3 stars! Short 10 minute episodes about cool microbes.
Obtain absorbance readings from these washes in the same way that was done for the flowthrough in Steps e-f
Th aim is get the column to have strong 260 absorbance due to oligonucleotides:
Keep adding lysis buffer until 260 absorbance is zero. This will take about 9-10 volumes
 
 
Nanodrop  2 uL "protein" at 280nm,
"Overlay spectra" between readouts.
Use lysis buffer for blank. Measure blank to makes sure there Nanodrop is clean and that there’s no preexisting absorbance.
Note: Looking for strong 260 peak (~0.85)
 
== Cloning and gene manipulation ==
===[[Commonly Used Plasmids]]===
===[[Plasmid Purification]]===
===[[Digest and Ligation]]===
===[[Creating Competent E. coli Cells]]===
===[[Transformation]]===
===[[Gibson Assembly]]===
===[[Creating Lac- E. coli Mutants]]===


== Phyllosphere protocols ==
== [[Waste Disposal]] ==


===[[Creating Sterile Agar Plates]]===
== [[Working with GitHub]]==
===[[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 ==
===[[Dual Layer Assays]]===
===[[Streptococcus DNA Extraction]]===
===[[Streptococcus Growth Curve Protocol]]===
===[[Streptococcus Growth Curve and Cell Count in Liquid Media]]===
===[[Log Phase Growth Curve and Cell Count in Liquid Media]]===


== ''Streptococcus suis'' protocols ==
== ''Streptococcus suis'' protocols ==
Line 229: Line 136:
===[[Measuring Competence : Fixation and Flow Cytometry]]===
===[[Measuring Competence : Fixation and Flow Cytometry]]===


== [[Interactions Protocols]] ==
== ''Arabidopsis thaliana'' protocols ==
===[[Zone of Inhibition Assay]]===


== [[Remote Molecular Biology]] ==
===[[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 ==
Line 242: Line 160:
=== [[expression]] ===
=== [[expression]] ===
=== [[lysis and immobilization]] ===
=== [[lysis and immobilization]] ===


==[[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"