Difference between revisions of "Team:Imperial College/Experiments"

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<li>For known plasmid: 10 & 100 ul of each transformation reaction onto a selection plate. For the rest of 890 ul:
 
<li>For known plasmid: 10 & 100 ul of each transformation reaction onto a selection plate. For the rest of 890 ul:
 +
<ol>
 
<li>Pellet cells at 8000rpm for 3 minutes</li>
 
<li>Pellet cells at 8000rpm for 3 minutes</li>
 
<li>Remove and dispense 600 ul of supernatant </li>
 
<li>Remove and dispense 600 ul of supernatant </li>

Revision as of 13:59, 16 October 2016

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
    • Incubate overnight at 37°C with plates upside down.