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Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize t...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361335/ https://www.ncbi.nlm.nih.gov/pubmed/25774528 http://dx.doi.org/10.1371/journal.pone.0119221 |
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author | Jacobus, Ana Paula Gross, Jeferson |
author_facet | Jacobus, Ana Paula Gross, Jeferson |
author_sort | Jacobus, Ana Paula |
collection | PubMed |
description | PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories. |
format | Online Article Text |
id | pubmed-4361335 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-43613352015-03-23 Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli Jacobus, Ana Paula Gross, Jeferson PLoS One Research Article PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories. Public Library of Science 2015-03-16 /pmc/articles/PMC4361335/ /pubmed/25774528 http://dx.doi.org/10.1371/journal.pone.0119221 Text en © 2015 Jacobus, Gross http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Jacobus, Ana Paula Gross, Jeferson Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli |
title | Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
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title_full | Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
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title_fullStr | Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
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title_full_unstemmed | Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
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title_short | Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli
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title_sort | optimal cloning of pcr fragments by homologous recombination in escherichia coli |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361335/ https://www.ncbi.nlm.nih.gov/pubmed/25774528 http://dx.doi.org/10.1371/journal.pone.0119221 |
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