Difference between revisions of "Team:Hong Kong-CUHK/Protocol"

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<br><br>
 
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<p style="font-family: roboto;font-size:120%;">Our team would like to share with you the protocols that we have been using over the years we used to generate our data and results. This library of protocols is introduced for the interest of reproducibility. </p>
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<p style="font-family: quicksand;font-size:120%;">Our team would like to share with you the protocols that we have been using over the years we used to generate our data and results. This library of protocols is introduced for the interest of reproducibility. </p>
  
 
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Revision as of 11:10, 22 August 2017



Our team would like to share with you the protocols that we have been using over the years we used to generate our data and results. This library of protocols is introduced for the interest of reproducibility.


  1. Thaw 100µL competent E. coli cells on ice for 10 minutes
  2. Add:
    • 10-20 µl DNA from a ligation reaction mix OR
    • 50-100ng DNA of a known plasmid
  3. Place the mixture on ice for 15 minutes.
  4. Heat shock at exactly 42°C for 90 seconds.
  5. Place on ice for 5 minutes.
  6. Pipette 900 µl of room temperature SOC or LB media into the mixture.
  7. Incubate at 37°C and 250 rpm for 60-120 minutes.
  8. Pellet cells at 13000rpm for 1 minutes
  9. Remove and dispense 600 µL of supernatant
  10. Re-suspend cells by light vortexing
  11. Plate resuspended cells as above
  12. Incubate overnight at 37°C with plates upside down.
  1. Prepare the buffers as follow:
  2. pH Composition
    2 0.2M KCl/HCl
    2 3M acetate buffer
    4l 0.5M MES buffer
    6 1M PIPES buffer/td>
    8 1M HEPES buffer
    10 0.2M NaHCO3/NaOH
    12 0.2M KCl/NaOH
  3. Diluted into buffers ranging in pH from 3.75–8.50 in 96-well plates.
  4. Determine absorbance/ fluorescence by Plate reader.