[Construction and identification of BKCaαsubunit expression plas-mid with double labeling of Flag and GFP]
Tao Li1, Xiu-Li Cheng2, Wen-Jun Huang1
1Key Laboratory of Medical Electrophysiology of Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease/Institute of Cardiovascular Research, Sichuan Medical University, Luzhou 646000.
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.
Related Concept Videos
Plasmids
Fixing Double-strand Breaks
Fixing Double-strand Breaks
Bode Plots Construction
Construction of Root Locus
For positive gain values, the root locus exists on the real axis to the left of an odd number of finite open-loop poles or zeros. The root locus starts at the open-loop poles and traces the paths of the closed-loop poles as the gain...
Construction of Frequency Distribution
First, make a table with two columns—one with the title of the data that needs to be organized, and the other column for frequency. [Draw a third column for tally marks if needed]. Then, take a look at the items given in the data set and decide if an ungrouped frequency distribution table or a grouped frequency distribution table would be more suitable. If there are large sets of different values, then it is...


