Team:Tec Guadalajara/Protocols

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Protocols

Synthesis of Graphene Oxide

1. Mix 3 g of graphite with 18 g of potassium permangante
2. Mix 360 mL of sulfuric acid with 40 mL of phosforic acid and let it cool down at 4 °C for 30 min
3. Combine both mixtures on an ice bath
4. Incubate at 50 °C, 700 rpm for 12 h
5. Let it cool down to room temperature and pour on 200 mL of distilled ice
6. Add 3 mL of hydrogen peroxide (35%)

This protocol is based on the work of Marcano et al (2010).

Reduction of Graphene Oxide

To obtain reduced graphene oxide we followed and made some modifications to the method described by Abdolhosseinzadeh, Asgharzadeh & Kim (2015).

Restriction Enzyme Digestion

1. Set up the following reaction

   Component	Quantity
   DNA	1 µg
   10X NEBuffer 2.1	5 µL (1X)
   EcoRI	1.0 µL (10 units)
   PstI	1.0 µL (10 units)
   Nuclease-free Water	to 50 µL

2. Incubate at 37 °C for 20 min
3. Incubate at 80 °C for 20 min

This protocol was adapted from NEBcloner™.

Ligation

1. Set up the following reaction in a microcentrifuge tube on ice

   Component	Quantity
   10X T4 DNA Ligase Buffer	2 µL (1X)
   Vector DNA	25 ng
   Insert DNA	60 ng
   T4 DNA Ligase	1.0 µL
   Nuclease-free Water	to 20 µL

2. Gently mix the reaction by pipetting up and down, and microfuge briefly
3. Incubate at 16 °C overnight or room temperature for 10 minutes
4. Incubate at 65 °C for 10 min to inactivate enzymes
5. Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells

This protocol was adapted from NEBcloner™. We also used NEBioCalculator™ the required insert mass.

1% Agarose Gel Electrophoresis

1. Gel Preparation
   1. Add 0.5 g of agarose to 50 mL of 1X TAE buffer
   2. Heat in microwave until the gel is homogeneous
   3. Let the gel cool down to 40 °C before adding 1 μL of SYBR Safe®
   4. Pour into a gel tray with the well comb in place
   5. Let the gel sit at room temperature until it solidifies completely
   6. Remove comb and place the gel into the gel box
   7. Fill the gel box with 1X TAE buffer
2. Sample Load
   1. Add SDS-containing loading buffer (6X) to the samples to a final concentration of 1X
   2. Incubate at 65 °C for 10 min
   3. Chill down on ice for 2 min
   4. Vortex gently and centrifuge
   5. Load molecular ladder into the gel's first lane
   6. Carefully load the rest of the samples
3. Gel Run
   1. Connect elecrodes and turn on the power source
   2. Run the gel at 90 V until the tracking dye is about 80% of the way down the gel
   3. Turn off the power source and disconnect the electrodes
   4. Transfer the gel from the gel box to a UV tray
   5. Visualize DNA using a gel imaging system

Bacterial Transformation

1. Thaw a tube of NEB 5-α competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µL of cells into a transformation tube on ice
2. Add 1-5 µL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex
3. Place the mixture on ice for 30 minutes. Do not mix
4. Heat shock at exactly 42 °C for exactly 30 seconds. Do not mix
5. Place on ice for 5 minutes. Do not mix
6. Pipette 950 µL of room temperature SOC into the mixture
7. Place at 37 °C for 60 minutes. Shake vigorously (300 rpm) or rotate
8. Warm selection plates to 37 °C
9. Mix the cells thoroughly by flicking the tube and inverting, then spread 10, 25, 50, and 75 µL in SOC
10. Incubate 8-12 hours to overnight at 37°C

This protocol was adapted from NEBCloner™.

Plasmid DNA Preparation

1. Isolate colonies and inoculate 4 mL of LB supplemented with chloramphenicol (50 ^μg⁄_mL)
2. Incubate with shaking for 12-18 h at 37 °C
3. Centrifuge at 5000 rpm for 3 min and discard the supernatant
4. Resuspend the pellet with 150 μL of solution 1. Transfer the solution into a new 1.5-mL microtube
5. Add 300 μL of solution 2 and mix by inversion. Incubate at room temperature for 5 min
6. Add 225 μL of solution 3 and mix by inversion. Incubate in ice for 5 min
7. Centrifuge at 14,000 rpm for 2 minutes and recover the supernatant
8. Add 800 μL of isopropanol, mix by inversion and incubate for 5 min in ice
9. Centrifuge at 14,000 rpm for 6 minutes and discard supernatant
10. Wash the pellet with ethanol (70%) and centrifuge at 14,000 rpm for 1 min
11. Discard the supernatant and dry the pellet
12. Resuspend DNA in 100 μL of Milli-Q water

BioBrick™ Submission

1. Sterilize the hood surface with 70% ethanol
2. Cover the 96 well plate with the provided plate lid (not with the adhesive foil)
3. Place the plate in the hood
4. With sufficient airflow, drying down 10 μL (25 ^ng⁄_μL) of purified plasmid DNA in the plate should take four hours
5. Once all wells have dried, cover the plate with the provided adhesive foil

We obtained this protocol from iGEM.