Chiba/Flagella/Making FliC-his
From 2007.igem.org
(Difference between revisions)
| Line 15: | Line 15: | ||
**Primer(FとR) 5μL | **Primer(FとR) 5μL | ||
**10×buffer 10μL | **10×buffer 10μL | ||
| - | **dNTPmix | + | **dNTPmix 10μL |
| - | **vent | + | **vent 1μL |
| - | **dH2O | + | **dH2O 68μL |
| Line 25: | Line 25: | ||
**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' | ||
| + | |||
| + | |||
| + | *pUC19-FliC vector とinsertオリゴをライゲーション. | ||
| + | ***[混ぜ表] | ||
| + | **insertオリゴ 2μL | ||
| + | **pUC19-FliC vector 2μL | ||
| + | **×5 Ligase Buffer 4μL | ||
| + | **Ligase 1μL | ||
| + | **dH2O 11μL | ||
| + | |||
| + | |||
| + | *GelCheck | ||
Revision as of 03:49, 27 October 2007
- pUC19 vectorにFliCをコードする遺伝子を挿入→pUC19-FliC(島田さんから)
- エキサイトPCR
- design insert(ヒスチジンタグ)
- pUC19-FliC vector とinsertオリゴをライゲーション.
- GelCheck
- エキサイト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]
- [混ぜ表]
- Template 1μL
- Primer(FとR) 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'
- pUC19-FliC vector とinsertオリゴをライゲーション.
- [混ぜ表]
- insertオリゴ 2μL
- pUC19-FliC vector 2μL
- ×5 Ligase Buffer 4μL
- Ligase 1μL
- dH2O 11μL
- GelCheck
