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Revision as of 14:28, 3 September 2007 (view source) Masahiro (Talk | contribs) ← Older edit		Revision as of 03:30, 5 September 2007 (view source) Masahiro (Talk | contribs) Newer edit →
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	#1,2をLigation '''done'''		#1,2をLigation '''done'''
	#Transformation '''done'''		#Transformation '''done'''
-	#Miniprep	+	#Miniprep '''done'''
	#Digestion test		#Digestion test
	*Check Protein		*Check Protein

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Revision as of 03:30, 5 September 2007

AHL TEAM

FliC TEAM

• pUC19-FliC-Hisの作製
  • LINKER LIGATION

1. pUC19-FliCをInverse PCR（五種類のF-Primer,R-Primer→15通りのPCR）⇒vectorの作成 done
   1. Check Size done
   2. Digestion(EcoRI,NcoI) done
   3. Zymo Clean(40μ溶出)→SAP（50μ系）→Zymo Clean（30μ溶出） done
2. Histag,Histag-antiをアニール done
   1. Digestion(EcoRI,NcoI) done
3. Ligation done
4. Transformation done
5. miniprep done
6. Digestion test done
7. Sequence→FliCに6×His配列が挿入されていることを確認 done

• RBSに問題有り→FliC領域の上流に開始コドンが二つ（RBSが結合する確率が高い）

1. 1のvectorにRBSを取り付ける done
2. 2-1産物をDigestion(HindⅢ,ApaⅠ) done
3. 1,2をLigation done
4. Transformation done
5. Miniprep done
6. Digestion test

• Check Protein

1. SDS-PAGE
2. Western-Bloting
3. Magnetic Beans