From 2007.igem.org

1. fliC is inserted in pUC19 vector→pUC19-FliC
2. ExSite PCR
3. design insert(6×Histidine-Tag)
4. Lagation pUC19-FliC vector and insert(flic+6×His-tag)
5. GelCheck

• ExSite PCR
• design primer
• Among the code of FliC, we selected five fragment codes from the outside part of the target filament, then designed each primers(207,228,240,252,267). Mounted pUC19-FliC to pUC19-FliC-vector.
  
  
• [Reaction Conditions]
  • Template 1μL
  • Primer(Foward and Reverse) 5μL
  • 10×buffer 10μL
  • dNTPmix 10μL
  • vent 1μL
  • dH2O 68μL

• design insert
  • six histidine loop peptide ("His-loop")
  • -Ser-Ser-His-His-His-His-His-His-Ser-Ser
  • encoded by the oligo 5'-TCT-TCT-CAT-CAT-CAT-CAT-CAT-CAT-TCC-TCC-3'
  • and anti oligo 5'-GGA-GGA-ATG-ATG-ATG-ATG-ATG-ATG-AGA-AGA-3'
  • restriction site (N termimal: EcoRⅠ,C terminal: NcoⅠ

• Ligation pUC19-FliC vector with insert.
• [Reaction Conditions]
  • insert 2μL
  • pUC19-FliC vector 2μL
  • ×5 Ligase Buffer 4μL
  • Ligase 1μL
  • dH2O 11μL

Fig. GelCheck showing pUC19-FliC-vector(3973bp).