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Background  Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one‐step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10–15 bp. The PCR products were then transformed into  E. coli  and cyclized by homologous recombination in vivo.    Methods  The pEGFP‐N1‐HA plasmid was constructed by one‐step PCR and transformation. Cells were transfected with pEGFP‐N1‐HA and pEGFP‐N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA‐GFP fusion protein was detected by confocal microscopy.    Results  The pEGFP‐N1‐HA plasmid was successfully constructed and HA expression in cells.    Conclusions  Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.    Key points   Significant findings of the study  A method to clone short DNA into plasmids was found.    What this study adds  Our study provides a flexible and economical option to clone short DNA into plasmids.

Cloning short DNA into plasmids by one‐step PCR