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Revision as of 04:48, 27 October 2007 (view source) Katabami (Talk | contribs) ← Older edit		Latest revision as of 04:52, 27 October 2007 (view source) Toyotaro (Talk | contribs)
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-	#FliC is inserted in pUC19 vector→pUC19-FliC	+	#fliC is inserted in pUC19 vector→pUC19-FliC
	#ExSite PCR		#ExSite PCR
	#design insert(6×Histidine-Tag)		#design insert(6×Histidine-Tag)
Line 7:		Line 7:
	*ExSite PCR		*ExSite PCR
-	**design primer	+	*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)	+	*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>
-	pUC19-FliCをpUC19-FliC-vectorに	+	*[Reaction Conditions]
-	***[PCR表]	+
-	***[Reaction Conditions]	+
	**Template 1μL		**Template 1μL
	**Primer(Foward and Reverse) 5μL		**Primer(Foward and Reverse) 5μL
Line 29:		Line 27:
	*Ligation pUC19-FliC vector with insert.		*Ligation pUC19-FliC vector with insert.
-	***[Reaction Conditions]	+	*[Reaction Conditions]
	**insert 2μL		**insert 2μL
	**pUC19-FliC vector 2μL		**pUC19-FliC vector 2μL
Line 37:		Line 35:
-	*GelCheck(3973bp)	+	[[Image:Gel_check.JPG]]<br>
-	[[Image:Gel_check.JPG]]	+	Fig. GelCheck showing pUC19-FliC-vector(3973bp).

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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

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