Team:UC Davis/Notebook/Protocols
From 2012.igem.org
Protocols
Registry Part Distribution Rehydration
- Add 20 µL sterile H2O to desired well in distribution plate.
- Incubate at room temperature for ~10 minutes.
- Transfer resuspension to microcentrifuge tube.
Transformations
Materials
- Competent Cells
- DNA template
- 800 µL LB
- LB+Antibiotic Plates
Procedure
- Thaw competent cells on ice.
- Transfer 50 µL of competent cells to chilled falcon tubes.
- Add 1 µL of template to cells (2.5 µL if dilute).
- Incubate on ice for 30 minutes.
- Heat schock in 42 °C water bath for 90 seconds.
- Immediately place back onto ice for 2 minutes.
- Add 800 µL of LB to each tube.
- Incubate at 37 °C for 1 hour.
- Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.
- Incubate overnight at 37 °C.
Restriction Enzyme Double Digest
Materials
- 22 µL dH20
- 1 µL BSA
- 5 µLBuffer
- 20 µL Template
- 1 µL Enzyme 1
- 1 µL Enzyme 2
Procedure
- Mix reactants, adding enzyme last.
- Place at 37 °C for 3-5 hours.
- If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
- Run on a gel and extract product.
PCR (sequence dependent)
Materials
- 10 uL Q Solution
- 5 µL 10x Buffer
- 1.25 µL 10mM dNTPs
- 1 µL Template
- 1 µL Forward Primer
- 1 µL Reverse Primer
- 0.3 µL Taq
- 0.15 µL PFU
- 30 µL dH20
Procedure
- Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
- Run in thermal cycler.
PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)
Materials
- 10 µL Q Solution
- 5 µL 10x Buffer
- 1.25 µL 10mM dNTPs
- 2.5 µL Forward Primer
- 2.5 µL Reverse Primer
- 0.3 µL Taq
- 0.15 µL PFU
- Template based on concentrations determined by SOEing calculator: “Link”
- ±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
Procedure
- Mix reactants into PCR tubes.
- Run in thermal cycler.
- Continue PCR SOEing of parts until completed.
Ligation
Materials
- Digested Vector
- Digested Insert
- Water
- T4 DNA Ligase
- T4 DNA Ligase Buffer
Procedure
- Mix these materials in the amounts determined by the reaction volume calculator here.
Ethylene Glycol Media
Materials
- 34 mM NaH2PO4
- 64 mM K2HPO4
- 20 mM (NH4)2SO4
- 1 µM FeSO4
- 0.1 mM MgSO4
- 10 µM CaCl2
- 30 mM Ethylene Glycol
- 30 mM Glycolate
- 0.5% wt/vol Casein Acid Hydrolysate
- 1.5% wt/vol Agar (if making solid media)
Procedure
- Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
- Mix together NaH2PO4, K2HPO4, (NH4)2SO4, FeSO4, MgSO4, and CaCl2.
- Titrate to pH 7 with HCl.
- Add the glycolate and casein acid hydrolysate.
- Mix in the ethylene glycol in a fume hood.
- If making solid media, also mix in agar.
- Autoclave on an appropriate cycle.
Ethylene Glycol Toxicity Assay
Materials
- Luria Broth Media
- Ethylene Glycol
- Tecan M200 Pro
- E. coli
Procedure
- Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
- Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
- Incubate at 37°C overnight on a shaker at 150 rpm.
- From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
- Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
- Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
- Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
- With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.
- Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
- Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.
- Aliquot the remaining dilutions as the diagram below depicts.
- Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
- Follow the steps of your Tecan machine appropriate to the specific bacterial strain.
EMS (Ethyl Methane Sulfonate Mutagenesis)
Materials
- Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
- K2HPO4: 10.5g
- KH2PO4: 4.5g
- (NH4)2SO4: 1g
- Sodium Citrate * 2H2O: 0.5g
- 1M Tris (pH 7.5): 200mL Tris
- EMS (Sigma)
- 50mL Conical Corning tubes
- 15mL Falcon tubes
- LB Broth
- Ethylene Glycol Agar Plates
Procedure
- 1. Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD 0.2.
- Chill the cells on ice and spin down the 10mL aliquots at max speed in Eppendorf Centrifuge 5810 R for 10 minutes.
- Wash twice with 10mL Buffer A.
- Pipet up and down to mix, pellet cells, decant supernatant.
- Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.
- Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells.
- Close tubes and parafilm the lid 2X.
- Vortex to mix and place in a secondary container (50mL conical tube).
- Transfer to mixing platform and agitate at ~30 rpm at 37°C.
- Withdraw samples at fixed time points.
- At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in Eppendorf Centrifuge 5810 R for 5 minutes.
- Discard supernatant in waste container.
- Wash twice in 5mL of Buffer A and discard supernatant in waste container.
- Re-suspend in 5mL of Buffer A and titer for viable cells.
- Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.
- Grow cultures overnight at 37°C and plate for mutants on EG agar plates.
- Look for viable cells
Safety: Health Hazards
- Acute toxicity (oral, dermal, inhalation), category 4
- Skin irritation, category 2
- Eye irritation, category 2
- Skin sensitization, category 1
- Specific Target Organ Toxicity – Single exposure, category 3
- LD50 – 470mg/kg
Recommended Safety & Handling
- Always work in fume hood and wear lab coat, goggles, and gloves.
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