Display options
Cite
Ramos AE, Muñoz M, Moreno-Pérez DA, et al. pELMO, an optimised in-house cloning vector. AMB Express. 2017;7(1):26doi: 10.1186/s13568-017-0324-2.
Ramos, A. E., Muñoz, M., Moreno-Pérez, D. A., & Patarroyo, M. A. (2017). pELMO, an optimised in-house cloning vector. AMB Express, 7(1), 26. https://doi.org/10.1186/s13568-017-0324-2
Ramos, Andrea E, et al. "pELMO, an optimised in-house cloning vector." AMB Express vol. 7,1 (2017): 26. doi: https://doi.org/10.1186/s13568-017-0324-2
Ramos AE, Muñoz M, Moreno-Pérez DA, Patarroyo MA. pELMO, an optimised in-house cloning vector. AMB Express. 2017 Dec;7(1):26. doi: 10.1186/s13568-017-0324-2. Epub 2017 Jan 24. PMID: 28116699; PMCID: PMC5265227.
Copy Download .nbib Format: NLM
Share it on

AMB Express. 2017 Dec;7(1):26. doi: 10.1186/s13568-017-0324-2. Epub 2017 Jan 24.

pELMO, an optimised in-house cloning vector.

AMB Express

Andrea E Ramos, Marina Muñoz, Darwin A Moreno-Pérez, Manuel A Patarroyo

Affiliations

  1. Molecular Biology and Immunology Department, Fundación Instituto de Inmunología de Colombia, Cra. 50 # 26-20, Bogotá, Colombia.
  2. PhD Programme in Biomedical and Biological Sciences, Universidad del Rosario, Bogotá, Colombia.
  3. Molecular Biology and Immunology Department, Fundación Instituto de Inmunología de Colombia, Cra. 50 # 26-20, Bogotá, Colombia. [email protected].
  4. School of Medicine and Health Sciences, Universidad del Rosario, Bogotá, Colombia. [email protected].

PMID: 28116699 PMCID: PMC5265227 DOI: 10.1186/s13568-017-0324-2

Abstract

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vector's multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the SmaI restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500 bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.

Keywords: Blunt-ended; Cloning vector; PCR cloning; Recombinant DNA technology; ccdB killer gene

References

  1. Biotechniques. 1996 Aug;21(2):320-3 - PubMed
  2. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2056-60 - PubMed
  3. Mol Microbiol. 1994 Mar;11(6):1151-7 - PubMed
  4. J Cell Commun Signal. 2008 Jun;2(1-2):25-45 - PubMed
  5. Biotechnol Appl Biochem. 2005 Dec;42(Pt 3):205-9 - PubMed
  6. Gene. 1996 Oct 17;176(1-2):23-6 - PubMed
  7. Biotechniques. 2000 Mar;28(3):400-2, 404, 406 passim - PubMed
  8. Crit Rev Biotechnol. 2002;22(3):225-44 - PubMed
  9. Plasmid. 2011 Sep;66(3):180-5 - PubMed
  10. Mol Biotechnol. 2011 Mar;47(3):229-33 - PubMed
  11. J Bacteriol. 1990 Nov;172(11):6557-67 - PubMed
  12. Sci Rep. 2012;2:1-8 - PubMed
  13. Protein Expr Purif. 2008 Jul;60(1):53-7 - PubMed
  14. J Mol Biol. 1992 Aug 5;226(3):735-45 - PubMed
  15. Anal Biochem. 2011 May 1;412(1):120-2 - PubMed
  16. Nucleic Acids Res. 1991 Mar 11;19(5):1156 - PubMed
  17. Biotechniques. 1997 Nov;23(5):938-41 - PubMed
  18. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642-6 - PubMed
  19. J Biol Chem. 1992 Jun 15;267(17):12244-51 - PubMed
  20. J Microbiol Methods. 2009 Jun;77(3):302-7 - PubMed
  21. Methods Mol Biol. 2002;192:111-9 - PubMed
  22. Gene. 2014 Jul 10;544(2):228-35 - PubMed
  23. Anal Biochem. 2012 Jun 15;425(2):166-8 - PubMed
  24. Gene. 2000 Mar 7;245(1):59-63 - PubMed
  25. Gene. 1995 Aug 30;162(1):159-60 - PubMed

Publication Types