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Revision as of 21:54, 1 November 2017
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Team
Team
Mentors
Collaborations
-
Project
Description
Interlab
Design
Experiments
Notebook
Contributions
Modeling
Results
Improve
Attributions
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Parts
Parts
Basic Parts
Composite Parts
- Safety
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Human Practices
Silver HP
Integrated and Gold HP
Public Engagement
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Judging
Medal Criteria
Judging Form
Transformations
BI21 Cells:
- Thaw 50 µL vial of BL21 cells on ice
- Inoculate 2µL DNA into 50µL BL21 cells
- incubate on ice for 30 minutes
- heat shock at 42℃ for 10 seconds
- incubate on ice for 5m
- Inoculate 250µL LB into vial
- incubate at 37℃ with shaking for 1.5 hours
- Plate 1x/9x
JM109 Cells:
- Thaw vial of JM109 cells on ice
- Label 14 mL Falcon tube & plane on ice
- Inoculate 50 µL JM109 cells into Falcon tube
- Inoculate 2µL DNA into 50µL JM109 cells
- incubate on ice for 20m
- heat shock at 42℃ for 45 seconds
- Inoculate 950µL LB into vial
- incubate at 37℃ with shaking for 1.5 hours
- Plate 1x/9x
Electrophoresis/Gel Protocols
1% Agarose Electrophoresis Gel
- Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask
- Heat in microwave for 35 seconds
- Swirl and heat in microwave for 25 seconds
- Swirl and heat in microwave for 15 seconds
- Swirl and heat in microwave for 7 seconds
- Swirl and ensure no ‘floaties’
- Cool outside of conical flask with water until ‘hand not’
- Pour into gel mold and add well comb
Gel Extraction
- Use ethanol-wiped scalpel to excise appropriate bands
- Mix and incubate at 50°C for 10 min
- Vortex every 2 mins until gel slice is completely dissolved
- Place nucleospin column into 2mL collection tube
- Add sample
- Centrifuge at 11,000 xg for 1 minute
- Discard throughflow and place column back in same tube
- Add 700 mL Buffer NT3
- Centrifuge at 11,000 xg for 1 minute
- Discard throughflow and place column back in same tube
- Repeat step 4
- Centrifuge at 11,000 xg for another minute
- To remove buffer
- Discard throughflow
- Centrifuge at 11,00 xg for add minute
- Place column in a clean, labelled 1.5 mL tube
- Add 25 mL prewarmed (50°C) Buffer NE
- Incubate at 50°C for 5 min
- Centrifuge at 50 xg for 1 minute
- Centrifuge at 11,000 xg for 1 minute
Plasmid Transformation Protocol
- Aliquot 1 ml media into 1.5 mL tubes
- Prechill labelled 14 mL round-bottom falcon tubes
- Add mL cell culture to tube
- Add appropriate amount of ligase rxh (2-2.5 L)
- Incubate in nice for 20 minutes
- Heat shock cells by placing at 42℃ for 45 seconds in waterbath
- Place on ice for 2 minutes
- Add 950 mL preheated media to each tube
- Incubate at 37℃ with 215 rpm shaking for 1.5 hr -> 20 minutes
Plating
- Plate on LB-antibiotic plates
- Plate 100 ml for 1x transformation
- Plate 100 mL 9x centrifuge remaining transformation at 2000 xg for 5 minutes and resuspended pellet in 100 L media
Plates Protocol
- Follow LB Agar Recipe
- Make sure to put stirring bar in water
- Needed to make into homogenous gel
- Needs to be sterile
- Put into Autoclave
- Lid should not be too tight
- Put in ice bath42°C
- Cool till not burning hand
- Put on stirring plate
- Put sterilized plates in Lamenia shield
- Using micropipette put .5 mL of chlor. And amp. agar solution
- 1000x dilution
- Amp. has to be defrosted in dark drawer
- Pour solution into plates
- Thin layer fill bottom
- Take lid slightly off to prevent condensation
QIA Prep Spin Mini Prep Kit
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- Example for 18 mL LB add
- 18 L20 mg/ L Kanamycin (antibiotic)
- 18 L 50 mg/ L ampicillin
- Aliquot 3.5 mL into labelled round bottom falcon tubes
- Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube
- Incubate overnight at 37°C with 215 rpm shaking
- To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube
- Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min
- Discard supernatant and repeat
- Resuspend pellet in Buffer P1 250 mL
- Add 250 L Buffer, P2 and mix by inverting 10x
- Add 350 L Buffer N3 and immediately mix by inverting 10x
- Centrifuge at 13, 200 rpm for 10 minute
- Pipet supernatant into labelled QIA prep spin column
- Centrifuge at 13, 200 rpm for 1 minute
- Discard thoroughflow
- Wash column by adding .5 mL Buffer PB *regular for low copy number plasmids*
- Centrifuge at 13, 200 rpm for 1 minute
- Discard thoroughflow
- Wash by adding .75 mL Buffer PE
- Centrifuge at 13, 200 rpm for 1 minute
- Discard thoroughflow
- Centrifuge for an addition minute to ensure removal of residual wash buffer
- Place spin column in a clean, labelled 1.5 mL tube
- Add 50 LEB to center of column & let stand for 1 minute at room temperature
- Centrifuge at 12,000 rpm for 1 minute
- Store at -20℃
