IGEM:MIT/2005/July wk 3:
Materials here: fluoroscein oligos primers for phoA, malE, scFvs, fecA, fecA', fecA promoter To Do: PCR -- malE fecA wt. fecA promoter scFvs (3) phoA Figure out: what is expected from the gels figure out general what to do for gels
Check PCRs products: run gels to check for size if pcr worked: digest malE, scFvs (gel extraction? or other method?) and put into plasmids as biobricks make plasmids of fecA and phoA if pcr doesn't work: PCR again --> troubleshoot Digest and assemble promoter and rbs combos for later assemblies
Materials here: primers: fecI, fecR To Do: Test if ligations worked: if yes: store biobricks of malE, scFvs transform E Coli with fecA/phoA plasmids if no: redo ligation PCR fecI PCR fecR wt.
Check PCR products: run gels to check for size if pcr worked: disgest fecI and put into plasmid as biobrick put fecR into plasmid to fix cut site if no: redo PCR PCR fecA/phoA plasmids with middle primers to fix cut sites
Check PCR products: gels for fecA/phoA after template plasmid has been removed--> cut with enzyme with newly removed cut site if PCRs worked: store fecA/phoA as biobricks Check ligations of fecI and fecR if ligations of fecI worked: store fecI as biobrick transform fecR into bacteria in order to remove pstI site