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===[[Propidium Iodide Staining on Agar Plates]]=== | ===[[Propidium Iodide Staining on Agar Plates]]=== | ||
===[[Spore Assay]]=== | ===[[Spore Assay]]=== | ||
1. Harvest overnight M. xanthus culture during log growth phase, wash twice in TPM, and concentrate to %x10^9 cells/ml. You will likely need several mL of culture to plate out a spore assay. Each large TPM plate will need 4 groups of 5 20uL spots (400uL per plate, where one plate = 1 replicate). You may wish to use the 15mL conical tubes and the centrifuge in superlab to prepare cells. | |||
2. While you are washing and preparing cells, leave TPM plates open to dry by flame or under the hood so that drying time is lessened. DO NOT leave flame unattended. | |||
3. Divide your plate into quadrants and in each quadrant, spot 5 20uL spots of cells at the proper density, just far apart enough so that your cell spots do not run into each other. Leave plates open until completely dry. | |||
4. Cover plates, invert them, and store in the incubator for 5 days at 32C. | |||
Day 2 (Read ahead to make sure you have the time set aside) | |||
1. Remove plates from the incubator and image if desired. | |||
2. Turn heat block to 50C in preparation, and check that you have enough sterile water to complete the below protocol. For each plate you prepared, you will need 9mL of sterile MiliQ water in a 15mL falcon tube (pre-filling and pre-labeling these tubes is recommended). | |||
3. Using a bent metal spatula (one should already be prepared for you in the lab, sterilize in between by dipping in 70% ethanol and running through the flame), gently scrape 3 of your 4 groups of cell spots from each plate together into one small area, careful so as not to disrupt the surface of the agar. Use the spatula to transfer the cells to the pre-filled 9mL of sterile water. | |||
4. Repeat step 3 for each of your plates. | |||
5. Important; WEAR EAR PROTECTION WHEN USING SONICATOR. Turn on the sonicator and sterilize the probe with isopropyl alcohol. Wipe excess with kimwipe and rinse in an empty tube of sterile water so there is no excess alcohol getting into your sample. | |||
6. With the sonicator set to an intensity of 18.5, and using a 'blank' tube of 9mL of water without cells, determine where the probe should be within your sample so that your output is 20 Watts for 10 seconds. Then use that same probe placement for all samples. Tip: use the gradations on the falcon tube for this, and try to keep your hand placement on the tubes consistent as well. | |||
7. Sonicate each sample at 20 Watts for 10 cycles each (1 cycle: 10 sec on, 30 sec off) for a total of 100 active seconds. This should deliver 2000J to your sample total. Sonicate just one sample at a time, sterilizing the probe with more isopropyl alcohol in between samples. | |||
8. Incubate samples at 50C for 2 hrs. | |||
9. While sonicated samples are incubating in the heat block, prepare your tubes of CTTSA. Each tube that you currently have will be split into three for technical replication. (Ex. if you have three tubes from the sonicator, you will dilute and plate those out on 9 total plates at the end. | |||
10. Melt down the CTTSA in the microwave, make 4mL aliquots for each tube that you need, and keep warm in a water bath or heat block until you need them. You can also prep tubes for serial dilution (see below), and take out the CTTYE plates you will need so they can come to room temperature. | |||
11. Once your samples are done heating, vortex to break up any clumps and you are ready to serial dilute. | |||
12. From your well-mixed tube of collected spores, take 100uL of sample into 900uL of sterile water, a 1:10 dilution. Repeat for another 1:10 dilution, changing tips in between and ensuring that samples are well mixed. Then transfer 10uL into 4mL of CTTSA and pour over a room temp CTTYE plate (with Kan if cells are resistant). [Note: three is potential that if we get the spore assay working well, another 1:10 dilution may be necessary, but start here] | |||
13. Allow CTTSA to solidify for 15min and put plates in incubator at 32C for 5 days. Count colonies and then back calculate from your serial dilution to determine the number of spores recovered. | |||
== Phage protocols == | == Phage protocols == |
Revision as of 13:53, 25 February 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
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
1. Harvest overnight M. xanthus culture during log growth phase, wash twice in TPM, and concentrate to %x10^9 cells/ml. You will likely need several mL of culture to plate out a spore assay. Each large TPM plate will need 4 groups of 5 20uL spots (400uL per plate, where one plate = 1 replicate). You may wish to use the 15mL conical tubes and the centrifuge in superlab to prepare cells.
2. While you are washing and preparing cells, leave TPM plates open to dry by flame or under the hood so that drying time is lessened. DO NOT leave flame unattended.
3. Divide your plate into quadrants and in each quadrant, spot 5 20uL spots of cells at the proper density, just far apart enough so that your cell spots do not run into each other. Leave plates open until completely dry.
4. Cover plates, invert them, and store in the incubator for 5 days at 32C.
Day 2 (Read ahead to make sure you have the time set aside)
1. Remove plates from the incubator and image if desired.
2. Turn heat block to 50C in preparation, and check that you have enough sterile water to complete the below protocol. For each plate you prepared, you will need 9mL of sterile MiliQ water in a 15mL falcon tube (pre-filling and pre-labeling these tubes is recommended).
3. Using a bent metal spatula (one should already be prepared for you in the lab, sterilize in between by dipping in 70% ethanol and running through the flame), gently scrape 3 of your 4 groups of cell spots from each plate together into one small area, careful so as not to disrupt the surface of the agar. Use the spatula to transfer the cells to the pre-filled 9mL of sterile water.
4. Repeat step 3 for each of your plates.
5. Important; WEAR EAR PROTECTION WHEN USING SONICATOR. Turn on the sonicator and sterilize the probe with isopropyl alcohol. Wipe excess with kimwipe and rinse in an empty tube of sterile water so there is no excess alcohol getting into your sample.
6. With the sonicator set to an intensity of 18.5, and using a 'blank' tube of 9mL of water without cells, determine where the probe should be within your sample so that your output is 20 Watts for 10 seconds. Then use that same probe placement for all samples. Tip: use the gradations on the falcon tube for this, and try to keep your hand placement on the tubes consistent as well.
7. Sonicate each sample at 20 Watts for 10 cycles each (1 cycle: 10 sec on, 30 sec off) for a total of 100 active seconds. This should deliver 2000J to your sample total. Sonicate just one sample at a time, sterilizing the probe with more isopropyl alcohol in between samples.
8. Incubate samples at 50C for 2 hrs.
9. While sonicated samples are incubating in the heat block, prepare your tubes of CTTSA. Each tube that you currently have will be split into three for technical replication. (Ex. if you have three tubes from the sonicator, you will dilute and plate those out on 9 total plates at the end.
10. Melt down the CTTSA in the microwave, make 4mL aliquots for each tube that you need, and keep warm in a water bath or heat block until you need them. You can also prep tubes for serial dilution (see below), and take out the CTTYE plates you will need so they can come to room temperature.
11. Once your samples are done heating, vortex to break up any clumps and you are ready to serial dilute.
12. From your well-mixed tube of collected spores, take 100uL of sample into 900uL of sterile water, a 1:10 dilution. Repeat for another 1:10 dilution, changing tips in between and ensuring that samples are well mixed. Then transfer 10uL into 4mL of CTTSA and pour over a room temp CTTYE plate (with Kan if cells are resistant). [Note: three is potential that if we get the spore assay working well, another 1:10 dilution may be necessary, but start here]
13. Allow CTTSA to solidify for 15min and put plates in incubator at 32C for 5 days. Count colonies and then back calculate from your serial dilution to determine the number of spores recovered.
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
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.