IGEM:MIT/2005/July wk 3:

Monday:

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

Tuesday:

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

Wednesday:

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.

Thursday:

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

Friday:

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