Chiba/Flagella/Making FliC-his
From 2007.igem.org
(Difference between revisions)
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**encoded by the oligo 5'-TCT-TCT-CAT-CAT-CAT-CAT-CAT-CAT-TCC-TCC-3' | **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' | **and anti oligo 5'-GGA-GGA-ATG-ATG-ATG-ATG-ATG-ATG-AGA-AGA-3' | ||
| - | ** | + | **restriction site (N termimal: EcoRⅠ,C terminal: NcoⅠ |
Revision as of 04:42, 27 October 2007
- FliC is inserted in pUC19 vector→pUC19-FliC
- ExSite PCR
- design insert(ヒスチジンタグ)
- Lagation of pUC19-FliC vector and insert
- 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")
- 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'
- restriction site (N termimal: EcoRⅠ,C terminal: NcoⅠ
- Ligation pUC19-FliC vector とinsertオリゴをライゲーション.
- [Reaction Conditions]
- insertオリゴ 2μL
- pUC19-FliC vector 2μL
- ×5 Ligase Buffer 4μL
- Ligase 1μL
- dH2O 11μL
- GelCheck(3973bp)
