Chiba/Flagella/Making FliC-his

From 2007.igem.org

(Difference between revisions)
Line 25: Line 25:
**encoded by the oligo 5'-TCT-TCT-CAT-CAT-CAT-CAT-CAT-CAT-TCC-TCC-3'  
**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'
**and anti oligo 5'-GGA-GGA-ATG-ATG-ATG-ATG-ATG-ATG-AGA-AGA-3'
 +
**制限酵素サイトN末端EcoRⅠ,C末端NcoⅠ
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-
*GelCheck
+
*GelCheck(3973bp)

Revision as of 04:15, 27 October 2007

  1. pUC19 vectorにFliCをコードする遺伝子を挿入→pUC19-FliC(島田さんから)
  2. エキサイトPCR
  3. design insert(ヒスチジンタグ)
  4. pUC19-FliC vector とinsertオリゴをライゲーション.
  5. GelCheck


  • エキサイト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]
      • [混ぜ表]
    • Template 1μL
    • Primer(FとR) 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'
    • 制限酵素サイトN末端EcoRⅠ,C末端NcoⅠ


  • pUC19-FliC vector とinsertオリゴをライゲーション.
      • [混ぜ表]
    • insertオリゴ 2μL
    • pUC19-FliC vector 2μL
    • ×5 Ligase Buffer 4μL
    • Ligase 1μL
    • dH2O 11μL


  • GelCheck(3973bp)
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