From 2007.igem.org

Revision as of 03:24, 27 October 2007 (view source) Risa (Talk | contribs) ← Older edit		Revision as of 03:26, 27 October 2007 (view source) Risa (Talk | contribs) Newer edit →
Line 11:		Line 11:
	[PCR表]		[PCR表]
	[混ぜ表]		[混ぜ表]
-	*Template1μL	+	*Template 1μL
-	*Primer(FとR)5μL	+	**Primer(FとR) 5μL
-	*10×buffer10μL	+	**10×buffer 10μL
-	*dNT	+	**dNTPmix
		+	**vent
		+	**dH2O
		+
		+
	*design insert		*design insert
	**six histidine loop peptide ("His-loop")		**six histidine loop peptide ("His-loop")

---

Revision as of 03:26, 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

• 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
  • vent
  • dH2O

• 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'