Chiba/Flagella/Making FliC-his

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< Chiba
Revision as of 04:44, 27 October 2007 by Katabami (Talk | contribs)
  1. FliC is inserted in pUC19 vector→pUC19-FliC
  2. ExSite PCR
  3. design insert(6×Histidine-Tag)
  4. Lagation of pUC19-FliC vector and insert
  5. GelCheck


  • ExSite PCR
    • design primer

FliCの可変領域のうち、フィラメントの外側部分から５か所を選び、プライマーを設計(207,228,240,252,267) pUC19-FliCをpUC19-FliC-vectorに

      • [PCR表]
      • [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")
    • アミノ酸配列-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 とinsertオリゴをライゲーション.
      • [Reaction Conditions]
    • insertオリゴ 2μL
    • pUC19-FliC vector 2μL
    • ×5 Ligase Buffer 4μL
    • Ligase 1μL
    • dH2O 11μL


  • GelCheck(3973bp)

Gel check.JPG

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