|
|
Line 12: |
Line 12: |
|
| |
|
| ==[[User:Madhvi Venkatesh|Madhvi Venkatesh]] 18:21, 10 February 2009 (EST)== | | ==[[User:Madhvi Venkatesh|Madhvi Venkatesh]] 18:21, 10 February 2009 (EST)== |
| Construction files for C and N terminal fliD parts:
| | So I rewrote |
| Note: fliD has an internal BamHI site
| |
| <pre>
| |
| Construction of {<fliD!} (N-terminal)
| |
| PCR Omv044F/Omv045R on MG1655 genomic DNA (485bp, gp=A)
| |
| PCR Omv045F/Omv044R on MG1655 genomic DNA (973bp, gp=B)
| |
| ----
| |
| PCR Omv044F/Omv044R on A + B (1435bp, EcoRI/BamHI)
| |
| Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI, 2974+910, L)
| |
| Product is pBca9495KC-M10000 {<fliD!}
| |
| ----
| |
| Omv044F Forward Bglbricking of {<fliD!} ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
| |
| Omv044R Reverse Bglbricking of {<fliD!} gcagtGGATCCttacttggaattactgttgttttcg
| |
| Omv045F Forward Removing BamHI site in {<fliD!} cgttaagcggCatccgtgatgcc
| |
| Omv045R Reverse Removing BamHI site in {<fliD!} ggcatcacggatGccgcttaacg
| |
| </pre>
| |
| | |
| <pre>
| |
| Construction of {<fliD>} (C-terminal type 1)
| |
| PCR Omv044F/Omv045R on MG1655 genomic DNA (485bp, gp=A)
| |
| PCR Omv045F/Omv046R on MG1655 genomic DNA (970bp, gp=B)
| |
| ----
| |
| PCR Omv044F/Omv046R on A + B (1432bp, EcoRI/BamHI)
| |
| Sub into pBca9495CA-Bca1144#5 (EcoRI/BamHI, 3039+910, L)
| |
| Product is pBca9495KC-M10001 {<fliD>}
| |
| ----
| |
| Omv044F Forward Bglbricking of {<fliD!} ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
| |
| Omv046R Reverse Bglbricking of {<fliD>} gcagtGGATCCcttggaattactgttgttttcg
| |
| Omv045F Forward Removing BamHI site in {<fliD!} cgttaagcggCatccgtgatgcc
| |
| Omv045R Reverse Removing BamHI site in {<fliD!} ggcatcacggatGccgcttaacg
| |
| </pre>
| |
| | |
| <pre>
| |
| Construction of {a~fliD>} (C-terminal type 2)
| |
| PCR Omv047F/Omv045R on MG1655 genomic DNA (492bp, gp=A)
| |
| PCR Omv045F/Omv046R on MG1655 genomic DNA (970bp, gp=B)
| |
| ----
| |
| PCR Omv047F/Omv046R on A + B (1439bp, EcoRI/BamHI)
| |
| Sub into pBca9495CA-Bca1144#5 (EcoRI/BamHI, 3039+910, L)
| |
| Product is pBca9495KC-M10002 {a~fliD>}
| |
| ----
| |
| Omv047F Forward Bglbricking of {a~fliD!} ccagtGAATTCatgAGATCTtcgcatggcaagtatttcatcgctggg
| |
| Omv046R Reverse Bglbricking of {<fliD>} gcagtGGATCCcttggaattactgttgttttcg
| |
| Omv045F Forward Removing BamHI site in {<fliD!} cgttaagcggCatccgtgatgcc
| |
| Omv045R Reverse Removing BamHI site in {<fliD!} ggcatcacggatGccgcttaacg
| |
| </pre>
| |