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Second Attempt - Protocol: | Second Attempt - Protocol: | ||
Thaw BL21(DE3) Cells on ice | #Thaw BL21(DE3) Cells on ice | ||
Pipette 50 μL of cells into transformation tube | #Pipette 50 μL of cells into transformation tube | ||
Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times | ##Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times | ||
170 ng/μl for R52-mCh-FL-BST | ##170 ng/μl for R52-mCh-FL-BST | ||
Unknown concentration for R52-mCh-H10-BST | ##Unknown concentration for R52-mCh-H10-BST | ||
163 pg/μl for pUC19 | 163 pg/μl for pUC19 | ||
Place mixture on ice for 30 mins. | Place mixture on ice for 30 mins. |
Revision as of 12:18, 5 September 2019
General microbiology protocols
Media Recipes
Reagent Recipes
Working with Antibiotics
Freezing -80 Stocks
Freezing Aliquots
Competition Assays
Generic PCR
Running DNA Gels
Cloning and gene manipulation
Commonly Used Plasmids
Plasmid Purification
Digest and Ligation
Creating Competent E. coli Cells
Transformation
Gibson Assembly
Phyllosphere 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
Streptococcus pneumoniae protocols
Dual Layer Assays
Streptococcus DNA Extraction
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
Remote Molecular Biology
Effect of Laboratory Protocols on Student Learning
Cambridge protocols
Storage buffer
transformation
Transformation of R5(2)-mCh-FL-BST and R5(2)-mCh-H10-BST
protocol:
- Thaw BL21(DE3) Cells on ice
- Pipette 50 μL of cells into transformation tube
- Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
- 170 ng/μl for R52-mCh-FL-BST
- Unknown concentration for R52-mCh-H10-BST
- Place mixture on ice for 30 mins.
- Begin to warm up LB Broth
- Heat shock at exactly 42°C for 10 secs.
- Used water bath
- Actual time: ~15 seconds
- Place on ice for 5 mins.
- Pipette 950 μL of room temperature LB Broth into mixture.
- Incubate at 37°C with 250 rpm for 60 mins.
- Warm selection plates to 37°C at the 30 min mark. (KAN resistance)
- Mix cells thoroughly and spread 100 μL of the mixture onto a plate
- One plate for either plasmid
- Leftover transformation mixtures stored in 4°C fridge
- Incubate overnight at 37°C
Results: The selection plates showed no colonies.
Troubleshoot: Increase heat shock time from 10 seconds to 45 seconds Additional transformation with pUC19 Plate the transformed cells with non-selection Agar plates along side selection plates Use heat block instead of water bath
Second Attempt - Protocol:
- Thaw BL21(DE3) Cells on ice
- Pipette 50 μL of cells into transformation tube
- Add 1 μL of protein encoded plasmids to transformation tube, flick 4-5 times
- 170 ng/μl for R52-mCh-FL-BST
- Unknown concentration for R52-mCh-H10-BST
163 pg/μl for pUC19 Place mixture on ice for 30 mins. Begin to warm up LB Broth Heat shock at exactly 42°C for 45 secs. Used heat block instead of water bath Place on ice for 5 mins. Pipette 950 μL of room temperature LB Broth into mixture. Incubate at 37°C with 225 rpm for 60 mins. Warm selection plates to 37°C at the 30 min mark. (KAN resistance) Mix cells thoroughly and spread 100 μL of the mixture onto a plate One Agar plate w/ KAN for BST plasmids One Agar plate w/ AMP for pUC19 One Agar plate w/o KAN for all 3 plasmids Leftover transformation mixtures stored in 4°C fridge Incubate overnight at 37°C
expression
lysis and immobilization
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