User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/02

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)

1. Gib_BBS F: 5'-actagtagcggccgctgcag (forward seq from the BB suffix)
2. 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

1. gg_BBS F: 5'-cgtctcactagtagcggccgctgcag (should also work for V0120)
2. gg_BBP R: 5'-cgtctctctagatgcggccgcgaattc (should also work for V0120)
3. gg_BBP-hPCD F: 5'-cgtctcCTAGAtggagctttcagcggtggg
4. gg_hPCD-BL01 R: 5'-cgtctcTCCATtctttccctttcctcaaagg
5. gg_BL01 F: 5'-cgtctcatggagctttcagcggtggg
6. gg_BL01-BBS R: 5'-cgtctcCTAGT