Team:Imperial College/Experiments

What should this page contain?
  • Protocols
  • Experiments
  • Documentation of the development of your project

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Materials:
LB broth
Ice
Selection plates

Methods:

  1. Thaw 50ul competent E. coli cells on ice for 10 minutes
  2. Add:
    • 5-10 µl DNA from a ligation reaction mix or
    • 10-100ng DNA of a known plasmid
  3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  4. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  6. Place on ice for 5 minutes. Do not mix.
  7. Pipette 950 µl of room temperature SOC or LB media into the mixture.
  8. Incubate at 37°C and 200-250 rpm for 60 minutes.
  9. Mix the cells thoroughly by flicking the tube and inverting.
  10. Spread:
    • For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 ul:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 ul of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
    • For known plasmid: 10 & 100 ul of each transformation reaction onto a selection plate. For the rest of 890 ul:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 ul of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
  11. Incubate overnight at 37°C with plates upside down.

Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner

  1. Add 5 ml liquid LB media into 12 ml culture tubes
  2. Add 5 μl of appropriate antibiotic into the broth
  3. Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
  4. Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm

Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA

Methods:
For a 25 µL reaction

  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
    Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine to begin thermocycling

For a 50 µL reaction
  1. In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
    Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity
  2. Gently mix the reaction
  3. If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  4. Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling

Thermocycling
Step Temperature (°C) Time
Initial denaturation 98 30 seconds
25-35 cycles 98 (denaturation)
45-72 (annealing) see Note 1
72 (extension)		 5-10 seconds
10-30 seconds
15-30 seconds per kb
Final extension 72 2-5 minutes
Hold 4 Indefinitely

Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/

Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube

Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner

  1. Add 5 ml liquid LB media into 12 ml culture tubes
  2. Add 5 μl of appropriate antibiotic into the broth
  3. Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
  4. Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm