Team:Hong Kong UCCKE/Protocols

Protocols

Restriction Enzyme Digestion
Materials needed
  • 70% ethanol
  • Paper towels
  • Ice
  • Container for ice
  • Lab marker/Sharpie
  • pSB1C3 linearized plasmid backbone (25ng/ul) (kit)
  • Part (25ng/ul)
  • 0.6ml tubes (kit)
  • 10X Red Buffer
  • NEB enzymes: EcoRI, PstI
  • Thermocycler, or waterbath and thermometer

Protocols
  1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
  2. Keep all enzymes and buffers used in this section on ice.
  3. Prepare a reaction mix by adding reagents in the order indicated in Table 1.
  4. Incubate at 37°C for 20 minutes.
Gel Electrophoresis
Materials needed
  • Agarose powder
  • TAE Buffer
  • Hot plate and Water Bath
  • SYBR Green dye
  • Gel Tank
  • DNA Samples
  • Loading Dye
  • 1KB ladder marker
  • Power supply

Prepare Agarose Gel
Gel solution (1%)
  1. Measure 0.6 g Agarose powder and add it to a 200 ml conical flask
  2. Add 60 ml TAE Buffer to the flask
  3. Cover the opening with aluminium foil
  4. Melt the agarose using the hot plate until the solution becomes clear
  5. Let the solution cool to about 50-55°C using water bath, swirl the flask occasionally to cool evenly.
  6. Add 6 ul of SYBR Green dye into the solution. (SYBR Green is light-sensitive, keep it dark)
  7. Seal the ends of the casting tray with two layers of tap
  8. Place the combs in the gel casting tray
  9. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white)
  10. Carefully pull out the combs and remove the tape
  11. Place the gel in the electrophoresis chamber
  12. Add enough TAE Buffer (About 120 ml) so that there is about 2-3 mm of buffer over the Gel

Loading The Gel
  1. Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - from what organism did the DNA came from, controls and ladder.
  2. Add 2 ul loading buffer on parafilm. (Same number as sample)
  3. Carefully pipette 10 ul of each sample and mix with loading buffer.
  4. Load the mixed sample to the corresponding well.
  5. Pipette 10 ul of the DNA ladder marker into at least one well of each row on the gel.

Running The Gel
1. Place the lid on the gel box, connecting the electrodes. 2. Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. 3. Turn on the power supply to about 120 volts, current:300 4. Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode. 5. Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA). 6. Let the power run until the blue dye approaches the end of the gel. 7. Turn off the power. 8. Disconnect the wires from the power supply. 9. Remove the lid of the electrophoresis chamber. 10. Using gloves, carefully remove the tray and gel.
Gel purification
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Ligation
Materials needed
  • T4 DNA Ligase Reaction Buffer
  • T4 DNA Ligase
  • Microcentrifuge
  • Thermocycler, or waterbath and thermometer
  • Restriction Digest: pSB1C3 linearized plasmid backbone
  • Restriction Digest: Insert

Protocol
  1. Add 2ul from the pSB1C3 linearized plasmid backbone digest.
  2. Add 3.3ul from the Part digest.
  3. Add 1ul of T4 DNA Ligase Reaction Buffer.
  4. Add 0.5ul of T4 DNA Ligase (keep this at -20°C until use!).
  5. Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.
  6. Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes using a thermocycler.
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