Difference between revisions of "Part:BBa K1638006:Design"
Jpettersen (Talk | contribs) (→Design Notes) |
Jpettersen (Talk | contribs) (→Design Notes) |
||
| Line 26: | Line 26: | ||
5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site. | 5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site. | ||
| − | 5.1) Forward: 5'- | + | 5.1) Forward: 5'-CGTCTGGAATTCGCGGCCGCTTCTAGAGNNN...NNN-3' (includes EcoRI and XbaI res. site) |
5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site) | 5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site) | ||
| Line 32: | Line 32: | ||
6) Run PCR with designed primers. | 6) Run PCR with designed primers. | ||
| − | 7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with | + | 7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with EcoRI and BamHI. |
8) Ligate backbone and PCR product | 8) Ligate backbone and PCR product | ||
Latest revision as of 15:56, 14 August 2015
10 aa linker with BamHI restriction site and TEV recognition site
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Standard assembly RFC[10] with this part creates scarsite (uacuag). Scarsites encodes stopcodons, which is inconvenient for the right use of this linker. The linker instead contains a 5'-end BamHI restriction site to be used in fusion to proteins with a 3'-end BamHI restriction site. For implementation, we recommend the following assembly method:
1) Design primers for amplification of the C-terminal fusion protein with the DNA sequence for the linker in the overhang of the forward primer.
1.2) Forward primer: 5'-CGCTTCTAGAGGGATCCGAAAATTTGTATTTTCAATCTGGTNNN...NNN-3' - (XbaI res. site and linker(with BamHI res. site) included)
1.3) Reverse primer: 5'-ATATCTGCAGCGGCCGCTACTAGTANNN...NNN-3' (SpeI and PstI res. site included)
2) Run PCR with designed primers.
3) Digest PCR-products and a standard backbone (e.g. pSB1C3) with XbaI and PstI
4) Ligate backbone and PCR product
Addition of linker_C-terminalFusionProtein to N-terminal fusion protein:
5) Design primers for amplification of N-terminal domain that includes a 3'-end BamHI res. site.
5.1) Forward: 5'-CGTCTGGAATTCGCGGCCGCTTCTAGAGNNN...NNN-3' (includes EcoRI and XbaI res. site)
5.2) Reverse: 5'-ATATGGATCCNNN...NNN-3' (includes BamHI res. site)
6) Run PCR with designed primers.
7) Digest PCR-products and plasmid containing linker_C-terminalFusionProtein with EcoRI and BamHI.
8) Ligate backbone and PCR product
Source
..