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 Karmella's BioBrick Cloning
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01/02/13
- Gibson assembly: order primers for vector pSB1A3
- Golden Gate assembly: order primers for pSB1A3 and Brady's parts
- Order enzyme for Golden gate assembly: BsmBI
Gibson Assembly design
- Previously tried to ligate Gibson insert into X/S-cut pSB1A3, unsuccessful. This time, PCR everything, including the vector, then do Gibson assembly.
- Retry assembly "hPCD-BL01" (see [1]). Make sure primer sequence overlaps with Brady's external sequences:
- BBP-hPCD fwd: gaattcgcggccgcttctaga-tggagctttcagcggtggg (first 21 = BioBrick prefix)
- BL01-BBS rev: ctgcagcggccgctactagt-gccaggatcccccgagcccc (first 20 = BioBrick suffix)
New primers designed to amplify pSB1A3 backbone (should also work for V0120)
- Gib_BBS F: 5'-actagtagcggccgctgcag (forward seq from the BB suffix)
- Gib_BBP R: 5'-tctagatgcggccgcgaattc (reverse seq from the BB prefix)
Golden Gate design
- Use BsmBI, per Dave Savage's recommendation
- cgtctc - 5bp overlap - part - 5bp overlap - gcagaga
- Forward - 5'-cgtctc NNNNN+20bp TOP
- Reverse - 5'-cgtctc nnnnn+20bp rev comp
New primers
- gg_BBS F: 5'-cgtctcactagtagcggccgctgcag (should also work for V0120)
- gg_BBP R: 5'-cgtctctctagatgcggccgcgaattc (should also work for V0120)
- gg_BBP-hPCD F: 5'-cgtctcCTAGAtggagctttcagcggtggg
- gg_hPCD-BL01 R: 5'-cgtctcTCCATtctttccctttcctcaaagg
- gg_
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Karmella's BioBrick Cloning
