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Fig. 2 | BMC Biotechnology

Fig. 2

From: A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends

Fig. 2

Gel electrophoresis separation of double-primer and single-primer PCR products. 1: Parental plasmid pET22b alone; 2: PCR product from reaction with primers pet22b1/pet22b2 using plasmid pET22b as template; 3: PCR product from reaction with primers radA1/radA2 using E. coli genomic DNA as template; 4: PCR product from reaction with primers GeneCluster3-1/GeneCluster3-2 using E. coli genomic DNA as template; 5: annealed PCR products from two single-primer linear reactions using primer pet22b3 or pet22b4, and the DNA sample from lane 2 as template; 6: annealed PCR products from two single-primer linear reactions using the primer radA2fw or radA2rv, and the DNA sample from lane 3 as template; 7: annealed PCR products from two single-primer linear reactions using the primer GeneCluster3-3 or GeneCluster3-4, and the DNA sample from lane 4 as template; 8: mixture of DNA samples from lanes 5 and 6 in a molar ratio of 1:3, ready for ligation; 9: mixture of DNA samples from lanes 5 and 6 in a molar ratio of 1:3, after ligation; 10: mixture of DNA samples from lanes 5 and 7 in a molar ratio of 6:1, ready for ligation; 11: mixture of DNA samples from lanes 5 and 7 in a molar ratio of 6:1, after ligation; 12: DNA ladder. PCR products were purified using a QIAquick purification kit (Qiagen) and electrophoresed in 1% agarose with Tris-acetate (40 mM Tris, 20 mM sodium acetate, 1 mM EDTA, pH 8.0) as the running buffer

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