Team:Copenhagen/Project/Experimental

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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 run a PCR

Purify with DNA purification kit

Cut with restrictionsenzymes EcoR1 and Pst1

Run on a gel and cut the wanted band out
http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf

Put it in the freezer

Cut the linarized plasmid backbone with Pst1 and EcoR1
http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones

Ligate CYP and Plasmid
http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones

Transform in XL1-Blue

Overnight cultures

Miniprep

Voila - you have a BioBrick