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IGEM team meeting notes 5 26 14
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= DNA Assembly Methods = Craig put together an awesome power point presentation: Powerpoint: https://docs.google.com/viewer?a=v&pid=forums&srcid=MDQ3ODI0ODE4NDM3NTM4NjIyMzUBMDM5OTQ5MTk5OTAwNDIzOTE2MjcBa0Z0ZTFkd2xycGdKATQBAXYy linkers == Traditional cloning == plasmid backbone: circular piece of DNA we're cloning into that has ab resistance, ORI, etc. http://help.teselagen.com/manual/section/8/ What RE sites can you use? RE sites can't be inside your DNA (will cute out GOI) Stoichiometry: important consideration when determining ideal plasmid backbone:GOI ratio If something goes wrong with ________ must use traditional cloning to fix it == Golden Gate Cloning == good for multiple sequence cloning you design DNA overhangs to ligate multiples pieces together in a strategic orientation no scar sequence introduced issues with scars occur when you're ligating together different protein domains same RE used for everything; restriction site is elminated from ligated product == BioBrick approach == use a few, specific restriction enzymes makes cloning easy if parts assembled on their own see http://parts.igem.org/Help:Standards/Assembly iGEM wants us to submit everything we do with BioBricks methods; different cloning methods are compatible with each other == SLIC: sequence and ligation independent cloning == one enzyme used for every reaction multi-step process; backbone vector contains sequences intrinsic to the plasmid backbone; GOI designed with same DNA at both ends throw in T4 DNA polymerase because it has 3' DNA exonuclease activity; eats up DNA from opposite ends of template strands e.coli (for example) repair machinery fills in gaps after ligation make constructs in e.coli and then express them in yeast == Gibson == three enzymes used for every reaction (more costly) one-step process Like SLIC except it uses T5 exonuclease (5' --> 3' activity; opposite of T4 used in SLIC) nicks repaired before they go into e. coli overhangs must lack secondary structure to avoid formation of hairpins which would compete with GOI for insertion into backbone only method with a theme song :) == GeneArt (not included in powerpoint) == Wrap-up & things to do: # creating cloning strategies on paper # make an excel sheet with all reagents; price break down; similar to spreadshet for DIY cloning class # update wiki with all work done individually or in groups; present updates to group meetings # finalize video and crowdtilt
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