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Revision as of 04:47, 27 October 2007 (view source) Toyotaro (Talk | contribs) ← Older edit		Revision as of 04:48, 27 October 2007 (view source) Katabami (Talk | contribs) Newer edit →
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	#ExSite PCR		#ExSite PCR
	#design insert(6×Histidine-Tag)		#design insert(6×Histidine-Tag)
-	#Lagation of pUC19-FliC vector and insert	+	#Lagation pUC19-FliC vector and insert(flic+6×His-tag)
	#GelCheck		#GelCheck

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Revision as of 04:48, 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

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")
  • -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

• GelCheck(3973bp)