Chiba/Flagella/Making FliC-his

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*pUC19 vectorにFliCをコードする遺伝子を挿入→pUC19-FliC
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#fliC is inserted in pUC19 vector→pUC19-FliC
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#design primer(エキサイトPCRのため)
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#ExSite PCR
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[PCR表]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)
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#design insert(6×Histidine-Tag)
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#エキサイトPCR→pUC19-FliC-vector
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#Lagation pUC19-FliC vector and insert(flic+6×His-tag)
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#design insert(ヒスチジンタグ)
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#GelCheck
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*six histidine loop peptide ("His-loop")
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*-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'
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#pUC19-FliC vector とinsertオリゴをライゲーション.
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*ExSite PCR
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[混ぜ表]
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*design primer
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#
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*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.<br><br>
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*[Reaction Conditions]
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**Template 1μL
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**Primer(Foward and Reverse) 5μL
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**10×buffer 10μL
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**dNTPmix 10μL
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**vent 1μL
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**dH2O 68μL
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 +
 
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*design insert
 +
**six histidine loop peptide ("His-loop")
 +
**-Ser-Ser-His-His-His-His-His-His-Ser-Ser
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**encoded by the oligo 5'-TCT-TCT-CAT-CAT-CAT-CAT-CAT-CAT-TCC-TCC-3'  
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**and anti oligo 5'-GGA-GGA-ATG-ATG-ATG-ATG-ATG-ATG-AGA-AGA-3'
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**restriction site (N termimal: EcoRⅠ,C terminal: NcoⅠ
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*Ligation pUC19-FliC vector with insert.
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*[Reaction Conditions]
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**insert 2μL
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**pUC19-FliC vector 2μL
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**×5 Ligase Buffer 4μL
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**Ligase 1μL
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**dH2O 11μL
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[[Image:Gel_check.JPG]]<br>
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Fig. GelCheck showing pUC19-FliC-vector(3973bp).

Latest revision as of 04:52, 27 October 2007

  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


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

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