Registry of standard biological parts/PCR-based assembly

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Demo

Going to get a better feel for this by doing a few biobrick constructions using PCR-assembly

  1. <bbpart>J04500</bbpart>+<bbpart>I13501</bbpart> = <bbpart>J04450</bbpart>
  2. <bbpart>R0010</bbpart>+<bbpart>I13504</bbpart> = <bbpart>J04430</bbpart>
  1. <bbpart>F2620</bbpart>+<bbpart>P1010</bbpart>+<bbpart>E0240</bbpart>
    • will try this one as a 3-way

UDG

Can use UDG (NEB USER enzyme) to replace all restriction digests and could also eliminate secondary PCRs also. You would just need to design your oligos to contain a U in the right location, assuming that there'll be a T somewhere in the vacinity of the ends of the parts you are trying to combine.

In-Fusion

References

  1. Lu Q. Seamless cloning and gene fusion. Trends Biotechnol. 2005 Apr;23(4):199-207. DOI:10.1016/j.tibtech.2005.02.008 | PubMed ID:15780712 | HubMed [Lu-2005]
  2. Hamilton MD, Nuara AA, Gammon DB, Buller RM, and Evans DH. Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res. 2007;35(1):143-51. DOI:10.1093/nar/gkl1015 | PubMed ID:17158165 | HubMed [Evans-2007]
  3. Horton RM, Cai ZL, Ho SN, and Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990 May;8(5):528-35. PubMed ID:2357375 | HubMed [Pease-1990]
  4. Yon J and Fried M. Precise gene fusion by PCR. Nucleic Acids Res. 1989 Jun 26;17(12):4895. PubMed ID:2748349 | HubMed [Fried-1989]
  5. Karreman C. Fusion PCR, a one-step variant of the "megaprimer" method of mutagenesis. Biotechniques. 1998 May;24(5):736, 740, 742. PubMed ID:9591118 | HubMed [Karrenman-1998]
  6. Wang HL, Postier BL, and Burnap RL. Optimization of fusion PCR for in vitro construction of gene knockout fragments. Biotechniques. 2002 Jul;33(1):26, 28, 30 passim. PubMed ID:12139251 | HubMed [Burnap-2002]
  7. Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 2007;35(7):e55. DOI:10.1093/nar/gkm106 | PubMed ID:17389646 | HubMed [Halkier-2007]
  8. Donahue WF, Turczyk BM, and Jarrell KA. Rapid gene cloning using terminator primers and modular vectors. Nucleic Acids Res. 2002 Sep 15;30(18):e95. PubMed ID:12235397 | HubMed [Jarrell-2002]
  9. Shevchuk NA, Bryksin AV, Nusinovich YA, Cabello FC, Sutherland M, and Ladisch S. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 2004 Jan 22;32(2):e19. DOI:10.1093/nar/gnh014 | PubMed ID:14739232 | HubMed [ladisch-2004]
  10. McDonald WF and Traktman P. Overexpression and purification of the vaccinia virus DNA polymerase. Protein Expr Purif. 1994 Aug;5(4):409-21. DOI:10.1006/prep.1994.1059 | PubMed ID:7950389 | HubMed [Traktman-1994]
All Medline abstracts: PubMed | HubMed