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===[[Measuring ''A. thaliana'' Phenotype using FIJI]]=== | ===[[Measuring ''A. thaliana'' Phenotype using FIJI]]=== | ||
===[[DNeasy PowerSoil Protocol]]=== | ===[[DNeasy PowerSoil Protocol]]=== | ||
'''Before you Begin:''' | |||
:Note: Upon arrival of the kit the CD2 solution should be stored at 2–8°C upon arrival. All other reagents and kit components should be stored at room temperature (15-25°C). | |||
'''Material:''' | |||
• Quick-Start Protocol for the DNeasy PowerSoil Pro Kit as published in the | |||
May 2019 DNeasy® PowerSoil® Pro Kit Guide as seen here www.qiagen.com/HB-2495. The safety data sheet can be found here: www.qiagen.com/safety. | |||
• Before starting ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing. If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves. Perform all centrifugation steps at room temperature (15-25°C). | |||
'''Protocol:''' | |||
1. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 mg of soil and 800 pl of Solution CD1. Vortex briefly to mix. | |||
2. Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5-2 ml tubes (cat. no. 13000-V1-24). Vortex at maximum speed for 10 min. Note: If using the Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5-10 min. | |||
:a. For more information about other bead beating methods, see the “Protocol: Detailed" section of DNeasy® Power Soil® Pro Kit Handbook. | |||
3. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min. | |||
4. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided). | |||
:a. Expect 500-600 pl. The supernatant may still contain some soil particles. | |||
5. Add 200 ul of Solution CD2 and vortex for 5 s. | |||
6. Centrifuge at 15,000 xg for 1 min at room temperature. Avoiding the pellet, transfer up to 700 ul of supernatant to a clean 2 ml Microcentrifuge Tube (provided). | |||
:a. Expect 500-600 ul. | |||
7. Add 600 ul of Solution CD3 and vortex for 5 s. | |||
8. Load 650 ul of the lysate onto an MB Spin Column and centrifuge at 15,000 x g for 1 min. | |||
9. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column. | |||
10. Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided). Avoid splashing any flow-through onto the MB Spin Column. | |||
11. Add 500 ul of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min. | |||
12. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube. | |||
13. Add 500 ul of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min. | |||
14. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided). | |||
15. Centrifuge at up to 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided). | |||
16. Add 50-100 ul of Solution C6 to the center of the white filter membrane. | |||
17. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications. | |||
:Note: We recommend storing the DNA frozen (-30 to -15°C or -90 to -65°C) as Solution Co does not contain EDTA. To concentrate DNA, please refer to the Troubleshooting Guide. | |||
== ''Streptococcus pneumoniae'' protocols == | == ''Streptococcus pneumoniae'' protocols == |
Revision as of 11:33, 16 July 2021
Lab Floor Plan (with list of materials)
General microbiology protocols
Media Recipes
Reagent Recipes
Working with Antibiotics
Freezing -80 Stocks
Freezing Aliquots
Competition Assays
Generic PCR
Gradient PCR
Running DNA Gels
Protein Purification
Sample Concentration
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
Arabidopsis protocols
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
DNeasy PowerSoil Protocol
Before you Begin:
- Note: Upon arrival of the kit the CD2 solution should be stored at 2–8°C upon arrival. All other reagents and kit components should be stored at room temperature (15-25°C).
Material:
• Quick-Start Protocol for the DNeasy PowerSoil Pro Kit as published in the May 2019 DNeasy® PowerSoil® Pro Kit Guide as seen here www.qiagen.com/HB-2495. The safety data sheet can be found here: www.qiagen.com/safety.
• Before starting ensure that the PowerBead Pro Tubes rotate freely in the centrifuge without rubbing. If Solution CD3 has precipitated, heat at 60°C until precipitate dissolves. Perform all centrifugation steps at room temperature (15-25°C).
Protocol:
1. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom. Add up to 250 mg of soil and 800 pl of Solution CD1. Vortex briefly to mix.
2. Secure the PowerBead Pro Tube horizontally on a Vortex Adapter for 1.5-2 ml tubes (cat. no. 13000-V1-24). Vortex at maximum speed for 10 min. Note: If using the Vortex Adapter for more than 12 preps simultaneously, increase the vortexing time by 5-10 min.
- a. For more information about other bead beating methods, see the “Protocol: Detailed" section of DNeasy® Power Soil® Pro Kit Handbook.
3. Centrifuge the PowerBead Pro Tube at 15,000 x g for 1 min.
4. Transfer the supernatant to a clean 2 ml Microcentrifuge Tube (provided).
- a. Expect 500-600 pl. The supernatant may still contain some soil particles.
5. Add 200 ul of Solution CD2 and vortex for 5 s.
6. Centrifuge at 15,000 xg for 1 min at room temperature. Avoiding the pellet, transfer up to 700 ul of supernatant to a clean 2 ml Microcentrifuge Tube (provided).
- a. Expect 500-600 ul.
7. Add 600 ul of Solution CD3 and vortex for 5 s.
8. Load 650 ul of the lysate onto an MB Spin Column and centrifuge at 15,000 x g for 1 min.
9. Discard the flow-through and repeat step 8 to ensure that all of the lysate has passed through the MB Spin Column.
10. Carefully place the MB Spin Column into a clean 2 ml Collection Tube (provided). Avoid splashing any flow-through onto the MB Spin Column.
11. Add 500 ul of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
12. Discard the flow-through and place the MB Spin Column back into the same 2 ml Collection Tube.
13. Add 500 ul of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
14. Discard the flow-through and place the MB Spin Column into a new 2 ml Collection Tube (provided).
15. Centrifuge at up to 16,000 x g for 2 min. Carefully place the MB Spin Column into a new 1.5 ml Elution Tube (provided).
16. Add 50-100 ul of Solution C6 to the center of the white filter membrane.
17. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream applications.
- Note: We recommend storing the DNA frozen (-30 to -15°C or -90 to -65°C) as Solution Co does not contain EDTA. To concentrate DNA, please refer to the Troubleshooting Guide.
Streptococcus pneumoniae protocols
Dual Layer Assays
Streptococcus DNA Extraction
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 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
Interactions Protocols
Zone of Inhibition Assay
Remote Molecular Biology
Effect of Laboratory Protocols on Student Learning
Interesting Podcats to Listen to When Doing Lab Work!
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.