Team:Copenhagen/Project/Experimental

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Revision as of 12:13, 11 July 2011


How to Mutate

  • Analysis have made you realise the need for mutations in the CYP in order to remove resctrictionsites
  • You design primers that fits your template but contains the altered base
  • You mix your primers with the template, dNTP, polymerase and HF buffer and run a non amplifying PCR
  • Digest the parental template DNA (which is methylated) with Dpn1 (which cut methylated DNA only)
  • Transform XL1-Blue competent cells with the mutated plasmid
How to make a BioBrick

  • Once you have a colony on your plate you take it out and place it in and overnight culture
  • You perform a Miniprep on your culture to purify your amplified plasmid
  • Digest your purified plasmid with restrictionsenzymes to check if your plasmids are mutated in the recognition site
  • Run the digest of the plasmid and hope to see a single band and not two (in the latter case your mutations failed)
  • Add prefix and suffix containing primers for the CYP in question and amplify with PCR
  • Purify with DNA purification kit
  • Cut with restrictionsenzymes EcoR1 and Pst1
  • Run on a gel and cut the wanted band out
  • Put it in the freezer
  • Cut the linarized plasmid backbone with Pst1 and EcoR1
  • Ligate CYP and Plasmid
  • Transform in XL1-Blue
  • Overnight culture
  • Hurra - You have a Biobrick
How to make a CyperMan

  • Cut the newly made Cyp BioBrick with restrictionsenzymes Xba and Pst1
  • Run on a gel and cut the wanted band out
  • Put it in the freezer
  • Cut the purified BioBrick J004500 ekspression vector from the kit with Spe1 and EcoR1
  • Ligate CYP and Vector
  • Transform in XL1-Blue
  • Overnight culture
  • Hurra - You have a CyperMan
Comments or questions to the team? Please

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