Chiba/Flagella/Making FliC-his
From 2007.igem.org
(Difference between revisions)
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*design primer | *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. | *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 | **Template 1μL | ||
**Primer(Foward and Reverse) 5μL | **Primer(Foward and Reverse) 5μL | ||
Revision as of 04:51, 27 October 2007
- fliC is inserted in pUC19 vector→pUC19-FliC
- ExSite PCR
- design insert(6×Histidine-Tag)
- Lagation pUC19-FliC vector and insert(flic+6×His-tag)
- 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
- GelCheck(3973bp)
