Abstract:
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.
journal_name
Thorac Cancerjournal_title
Thoracic cancerauthors
Tao CC,Yang Y,Li F,Qiao L,Wu Y,Sun XD,Zhang YY,Li CLdoi
10.1111/1759-7714.13660subject
Has Abstractpub_date
2020-11-01 00:00:00pages
3409-3415issue
11eissn
1759-7706issn
1759-7714journal_volume
11pub_type
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