Objective: This study aimed to construct a large conductance calcium activated potassium channel α (BKCa) subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study.
Methods: Based on the existing coding BKCa channel α subunit expression plasmid pcDNA3.1-hSlo, we constructed a double-tag expression plasmid, namely, pcDNA3.1-Flag-hSlo-GFP (Flag-hSlo-GFP).
Results: Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit, and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful.
Conclusions: The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.
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| http://dx.doi.org/10.13459/j.cnki.cjap.2016.03.024 | DOI Listing |
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