Team:Copenhagen/Project/Experimental

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 Mail