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


== Laboratory information ==
=== [[Detailed Lab Task Descriptions]] ===
 
 


== General microbiology protocols ==
== 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]]===


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


===Creating Sterile Agar Plates===
===[[Creating Lac- E. coli Mutants]]===
====Version 1: Copied from ABRC's Seed Handling FAQ for Seed Handling====
'''Citation: ABRC Seed Handling FAQ PDF [https://abrc.osu.edu/sites/abrc.osu.edu/files/abrc_handling_seed_2013.pdf], accessed Sept. 23, 2018.'''


#Add 4.31 g of Murashige and Skoog (MS) basal salt mixture and 0.5 g of 2-(N-Morpholino) ethanesulfonic acid (MES) to a beaker containing 0.8 L of distilled water and stir to dissolve. Add distilled water to final volume of 1 L. Check and adjust pH to 5.7 using 1M KOH.
#Divide the media into two 1 L glass bottles, 500 mL in each. Add 5 g of agar granulated per bottle. Keep the lid loose.
#Autoclave for 20 min at 121°C, 15 psi with a magnetic stirring device in the bottle.
#Place the bottles on a stir plate at low speed, and allow the agar medium to cool to 45-50°C (until the container can be held with bare hands).
#Starting from this step, perform all the steps in sterile conditions in a laminar flow hood. Add (optional) 1-2% sucrose and 1 mL Gamborg’s Vitamin Solution, stirring to evenly dissolve. Optional sucrose and vitamins should be added after autoclaving and only after the agar media cools, because vitamins are thermo-labile and 15-25% of the sucrose may be hydrolyzed to glucose and fructose at elevated temperatures. Plants grow more vigorously and quickly on media containing 1-2% of sucrose, however, fungal and bacterial contamination must be rigorously avoided by seed sterilization. Note that germination of some mutants might be delayed on sucrose-containing media.
#Label the bottom of Petri plates with identification number or name, including the date.
#Pour enough media into plates to cover approximately half of the depth of the plate.
#Allow the plates to cool at room temperature for about an hour to allow the agar to solidify. If the plates are not to be used immediately, wrap them in plastic and store at 4°C (refrigerator temperature). Covered plates, boxes, or tubes with solidified agar can be stored for several weeks at 4°C in a container that prevents desiccation.
##NOTE: Our lab will likely be using Magnenta GA-7 plant culture boxes rather than petri dishes - if any changes to this protocol are necessary due to this difference, it will be addressed in Version 2 of this protocol.


===Sterilization and Germination Protocol for ''Arabidopsis thaliana'' Seeds in Gnotobiotic Experiments===
== ''Streptococcus pneumoniae'' protocols ==
====Version 1: Adapted from ABRC's Seed Handling FAQ for Seed Handling====
===[[Dual Layer Assays]]===
 
===[[Streptococcus DNA Extraction - Genome Prep]]===
Citation: ABRC Seed Handling FAQ PDF [https://abrc.osu.edu/sites/abrc.osu.edu/files/abrc_handling_seed_2013.pdf], accessed Sept. 23, 2018.
 
# Surface sterilize seeds in microcentrifuge tubes by soaking for 20 min in 50% household bleach with the addition of 0.05% Tween®20 detergent.
# Remove all bleach residue by rinsing 5-7 times with sterile distilled water.
## Add 1 mL of sterile distilled water to epitube and invert once; remove water and continue 5 more times.
#For planting of individual seeds at low density, adhere one seed to the tip of a pipette using suction, then release seed onto the agar in desired location. For planting seeds at higher densities, mix seeds in sterile distilled water (or 0.1% cooled top agar), pour onto plate, and immediately swirl to achieve even distribution. Use a sterile pipet tip to adjust the distribution and remove excess water. Allow the water or top agar to dry slightly before placing lid onto plate.
# Seal with Micropore paper tape to prevent desiccation, while allowing slight aeration.
# Place the plates at 4°C for 3 days. This cold treatment, also called stratification, will improve the rate and synchrony of germination. The use of an extended cold treatment of approximately 7 days is especially important for freshly harvested seeds, which have more pronounced dormancy. An extended cold treatment is also necessary for certain natural accessions (e.g., Dobra-1, Don-0, Altai-5, Anz-0, Cen-0, WestKar-4). Cold treatment of dry seeds is usually not effective in breaking dormancy.
## NOTE: Experiments will be conducted that will determine what amount of cold stratification leads to maximum synchrony in seed germination time for Col-0 ''Arabidopsis thaliana'' seeds.
 
===Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments===
====Version 1: Adapted from ABRC Seed Handling FAQ PDF [https://abrc.osu.edu/sites/abrc.osu.edu/files/abrc_handling_seed_2013.pdf], accessed Sept. 23, 2018.====
 
# Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil [https://sites.cns.utexas.edu/greenhouse/arabidopsis-growing-protocol (see Arabidopsis Growing Protocol)] pre-moistened with distilled water. Do not compact soil.
# Add 1 mL of sterile distilled water to epitube containing the seeds to be planted. Invert and allow seeds to absorb water and increase in size slightly. Remove water with pipette and dispose.
# Using a pipette tip, take one seed and place it on the surface of the soil in one seed flat cell. Repeat for how many seeds are to be planted.
# Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
# Cover the seed flat(s) with perforated plastic wrap that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with  0.5 - 1 mL of sterile distilled water if soil dries out.
# Place seed flats in greenhouse after cold stratification time period is completed.
## Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.
 
====Version 2: Non-sterile Germination of ''Arabidopsis thaliana'' Seeds Using '''Folded Paper''' for Seed Dispersal====
 
:Note: This protocol was created as a variation on Version 1 because of issues encountered during the first planting of seeds, namely the difficulty of using a pipet to transfer seeds when there is no viscous vehicle for this action.
 
# Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil [https://sites.cns.utexas.edu/greenhouse/arabidopsis-growing-protocol (see Arabidopsis Growing Protocol)] pre-moistened with distilled water. Do not compact soil.
# Fold a piece of printer paper in half to create a strong valley fold. Tap approximately 25 seeds from the seed epitupe onto the paper, and shake the paper slightly so that the seeds align with the crease.
# Gently tap the paper so that at least on seed lands on the surface of the soil in one seed flat cell. Repeat so that 25 cells are planted with seeds.
## Unfortunately, the small size of the seeds and imprecise nature of the dispersal means that sometimes a few seeds will land in the cells, rather than just one.
# Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
# Cover the seed flat(s) with perforated aluminum foil that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with  0.5 - 1 mL of sterile distilled water if soil dries out.
# Place seed flats in greenhouse after cold stratification time period is completed.
## Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.
 
====Version 3: Non-sterile Germination of ''Arabidopsis thaliana'' Seeds Using '''Folded Paper''' for Seed Dispersal====
 
:Note: This protocol was created as a variation on Version 2 because it did not specify that the tin foil had to be removed after placing seed flats in greenhouse, and it did not give greenhouse irrigation instructions.
 
# Obtain clean seed flats (6x12 cells, 2 in. deep) and fill with a uniform amount of Promix sterile potting soil [https://sites.cns.utexas.edu/greenhouse/arabidopsis-growing-protocol (see Arabidopsis Growing Protocol)] pre-moistened with distilled water. Do not compact soil.
# Fold a piece of printer paper in half to create a strong valley fold. Tap approximately 25 seeds from the seed epitupe onto the paper, and shake the paper slightly so that the seeds align with the crease.
# Gently tap the paper so that at least on seed lands on the surface of the soil in one seed flat cell. Repeat so that 25 cells are planted with seeds.
## Unfortunately, the small size of the seeds and imprecise nature of the dispersal means that sometimes a few seeds will land in the cells, rather than just one.
# Water each cell with approximately 0.5 - 1 mL of sterile distilled water, being careful not to wash away the seed.
# Cover the seed flat(s) with perforated aluminum foil that does not touch the surface of the soil. Place seed flat(s) in 4°C cold room for cold stratification. Check on seed flat(s) daily and water with  0.5 - 1 mL of sterile distilled water if soil dries out.
# Place seed flats in greenhouse after cold stratification time period is completed - remove tin foil
# Water seed flats every day before noon for about 30 seconds with a general garden hose mister attachment.
## Our experiment will place seeds in the cold room at 8, 6, 4, and 2 days out from when they will be placed in the greenhouse. This will allow us to determine what amount of time is best for encouraging germination synchrony of seeds.
 
====Growth Stage Phenotype Definitions====
 
:Note: This information is taken ''directly'' from Table 1 of the paper [http://www.plantcell.org/content/13/7/1499  Growth Stage–Based Phenotypic Analysis of Arabidopsis]. This information is to be used to consistently discuss the growth stage phenotypes of ''Arabidopsis thaliana'' plants for all parts of their life cycle.
 
Col-0 Data
 
{| class="wikitable"
|-
! Stage
! Description
! Days
|-
| 1.0
| cotyledons fully opened
| 6.0
|-
| 1.02
| 2 rosette leaves >1 mm
| 10.3
|}
 
:Note: the descriptor "Days" gives the number of days after planting, '''given a 3 day cold stratification period.'''


More information will be added to this table from the paper as time goes by.
===[[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)]]===


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


===[[Streptococcus mutans Transformation Media Recipe (SMUR)]]===


====''Temperature''====
== ''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]]===


Grow chamber temperatures are kept as close to 22°C as possible.
===[[Rehydrating New Primers]]===


: Note: The shelves used to grow gnotobiotic ''Arabidopsis thaliana'' plants are enclosed by a mylar and fabric cover that helps to insulate against changes in temperature, and to raise the internal temperature slightly above the 18°C temperature of the grow room.
===[[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]]===


:''Arabidopsis thaliana'' plants grown in non-sterile environments will be in the greenhouse, which has an internal temperature that is slightly higher and more stable than that of the surrounding external atmosphere. Although there will be more temperature fluctuation in this location, the plants will be exposed to identical conditions when they are transferred outdoors.
== Phage protocols ==
===[[Media and Passaging]]===
===[[Plaque Assays with Soft Agar]]===
===[[Serial Dilutions of Phage]]===
===[[Calculating Virus Titre]]===
===[[Measuring Burst Size]]===


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


Lighting should be around 100-200 umol/m2/s during the 16 hour photoperiod.
== [[Remote Molecular Biology]] ==


: Note: Our grow chamber is equipped with sets of 4 flourescent bulbs per shelf - each bulb is 2650 Lumen, and so 4 of them in conjunction equals 10600 lumen. Based on the size of our shelves, that is roughly equivalent to 10600 lux (as each of our shelves are around 1 square meter). A conversion website [https://www.apogeeinstruments.com/conversion-ppfd-to-lux/] shows that 100-200 umol/m2/s is equivalent to 7,400 and 14,800 lux, respectively. Thus, our approx. 10,600 Lux light source falls within the necessary range of lighting.


====''Irrigation''====
== [[Effect of Laboratory Protocols on Student Learning]] ==
== Interesting Podcasts to Listen to When Doing Lab Work! ==


Irrigation is only needed for Arabidopsis thaliana plants grown in non-sterile conditions. Once seed flats have been transferred to the greenhouse, keep soil well watered until true leaves have appeared on seedlings - at this point, "reduce the watering frequency to as low as once or twice per week as needed" [https://abrc.osu.edu/sites/abrc.osu.edu/files/abrc_handling_seed_2013.pdf]. Watering can be conducted by misting the plants for 30 seconds with a hose mister.
*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.  


===Inoculation of ''Arabidopsis thaliana'' with Microbes===
*This Week in Virology
**By Vincent Racaniello


:True leaves of young ''Arabidopsis thaliana'' can be inoculated via either a foliar spray [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247453/] or pipetting [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126462/] of the microbial suspension. A foliar spray would likely coat the leaves more evenly and effectively, but it would also likely inoculate the agar as well, potentially leading to adverse bacterial growths on the agar surface. Pipetting would be more labor intensive and possibly less effective at leaf inoculation, but it would ensure that ''only'' the plant received the microbial suspension.
*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.  


====''Foliar pipetting''====
*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.


:Note: these steps are to be observed under sterile conditions.
*Journey to the Micro Cosmos
# Obtain a suspension of phyllosphere microbiota - this can be achieved by following steps one and two in [[Removal and DNA Extraction of Phyllosphere Microbes]] and then removing the leaves from the mixture.
**3 stars! Short 10 minute episodes about cool microbes.
# Dilute cell suspension so that "OD600 = 0.02" [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126462/]
# Pipet "5 to 10 uL" of cell suspension onto the leaves "when plants [are] 2 weeks old."
# For control plants that will receive no bacteria, pipet "sterile 10 mM MgCl2 solution to plant ... leaves."


====''Foliar spray''====
== [[Waste Disposal]] ==
 
:Follow the same steps as for [[Foliar pipetting]], but instead of using a pipet, apply 5 to 10 uL of cell suspension using a small 30 mL spray bottle.
 
===Removal and DNA Extraction of Phyllosphere Microbes===
 
: This protocol based directly on A Direct Method to Isolate DNA from Phyllosphere Microbial Communities Without Disrupting Leaf Tissues. W Suda, M Oto, S Amachi, H Shinoyama, M Shishido. Microbes Environ. Vol. 23, No. 3, pg. 248-252, 2008. [https://www.jstage.jst.go.jp/article/jsme2/23/3/23_8516/_pdf/-char/en] Minor changes were made to the tenses and placements of verbs for protocol clarity.
 
# Place 5 g non-shredded fresh leaf sample in a 50 mL sterilized polypropylene tube. Add 5 mL of extraction buffer (100 mM Tris-HCl, pH 9.0, 40 mM EDTA), 1 mL of 10% SDS, and 3 mL of benzyl chloride.
# Incubate the tube for 15 min at 50°C by mixing repeatedly with 1-min intervals so that the two phases are thoroughly mixed.
# Remove leaves from the tube, and add 3 mL of 3M sodium acetate (pH5.0).
# Incubate on ice for 10 min, then centrifuge the suspension (6000xg, 15 min, 4°C).
# Transfer the aqueous phase to a new centrifuge tube, and precipitate DNA by adding an equal volume of isopropanol followed by centrifugation (9000xg, 15 min, 4°C).
# Air-dry pellet, then resuspend in 200 uL of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0).
 
===ARISA===
 
====''Primers''====
 
Use "primers ITSF (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITSReub (5′-GCCAAGGCATCCACC-3′)", which are, " respectively, complementary to positions 1423 and 1443 of the 16S rRNA and 38 and 23 of the 23S rRNA of E. coli." [https://aem.asm.org/content/70/10/6147]
 
====''ARISA Protocol''====
 
=====Version 1=====
:Note: this protocol is Copied from https://academic.oup.com/femsec/article/85/3/483/583834#95229670  Diversity of the skin microbiota of fishes: evidence for host species specificity.
 
#Extracted DNA was used as a template for PCR on the internal transcribed spacer region using the ITS-FEub (5′-GTCGTAACAAGGTAGCCGTA-3′) and ITS-REub (5′-GCCAAGGCATCCACC-3′) primers (Cardinale et al., 2004).
#Ribosomal internal spacer analysis (RISA) was performed as previously described by Arias et al. (2006) with the following modifications. The PCR mix contained 1× Taq buffer, 0.4 mM dNTPs (Promega, Madison, WI), 0.4 μM ITS-FEub primer, 0.2 μM ITS-R primer, 0.02 μM ITS-REub labeled primer, 5 mM MgCl2, 1 U of Taq polymerase (5 PRIME, Inc., Gaithersburg, MD), and 100 ng of template DNA in a final volume of 50 μL.
# PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 55 °C for 1 min, and 68 °C for 2 min, ending with a final extension at 68 °C for 7 min.
# Ten microliters of each PCR product was diluted with 5 μL AFLP® Blue Stop Solution (LI-COR). Diluted samples were denatured at 95 °C for 5 min followed by rapid cooling prior to gel loading to prevent reannealing.
#PCR products were electrophoresed on the NEN Global Edition IR2 DNA Analyzer (LI-COR) following manufacturer's instructions. One microliter of sample was loaded into each well.
 
 
Sources:
https://academic.oup.com/femsec/article/85/3/483/583834#95229670  Diversity of the skin microbiota of fishes: evidence for host species specificity
https://aem.asm.org/content/70/10/6147 Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities
 
== ''Streptococcus pneumoniae'' protocols ==


== [[Working with GitHub]]==




== ''Streptococcus suis'' protocols ==
== ''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 ==
== 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"