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	*GelCheck(3973bp)		*GelCheck(3973bp)
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Revision as of 04:26, 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)