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Pipette tips (Axygen, catalog numbers: T-300, T-200-Y, T-1000-B )
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Scalpel blade (GLOBALMED, catalog number: BD001 )
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1.5 ml microcentrifuge tubes (Axygen, catalog number: MCT-150-C )
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Petri dish (Nunc, catalog number: 150466 )
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24-well plate (Nunc, catalog number: 142475 )
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Capillary glass (Sutter Instrument Company, catalog number: B100-58-10 )
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Parafilm (Bemis, catalog number: PM-996 )
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Micropipette tip (SUTTER, catalog number: BF100-50-10 )
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3-ml plastic pipettes dropper ( KEHUA, catalog number: KH145 )
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0.22 μm Syringe filter (GREEN MALL, catalog number: RSF02 )
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Competent E. coli DH5α (TIANGEN, catalog number: CB101-02 )
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pT7TS (provided by Xiaorong Fan, Nanjing Agricultural University)
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Plasmid Purification Midi Kit (QIAGEN, catalog number: 12143 )
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KOD polymerase (TOYOBO, catalog number: KOD-201B )
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Agarose (BIOWEST, catalog number: B21033 )
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DNA Gel Extraction Kit (AxyGEN, catalog number: AP-GX-250 )
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Restriction enzyme Bgl II (Takara Bio, catalog number: 1021A )
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Restriction enzyme Spe I (Takara Bio, catalog number: D1086a )
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Restriction enzyme BamH I (Takara Bio, catalog number: 1010 )
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T4 Ligase (STRATAGENE, catalog number: M0202V )
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Plasmid DNA Extraction Mini Kit (Axygen, catalog number: AP-MN-P-250 )
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Plasmid purification Midi (QIAGEN, catalog number: 12145 )
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AxyPrep DNA Gel Extraction Kit (Axygen, catalog number: AP-GX-250 )
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Isopropanol (SCRC, catalog number: 80109218 )
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Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
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Proteinase K (Sigma-Aldrich, catalog number: P2308 )
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Chloroform (SCRC, catalog number: 100068720 )
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mMESSAGE mMACHINE® Kit High Yield Capped RNA Transcription Kit (Ambion, catalog number: AM1344 )
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Xenopus (sh-maosheng Co.)
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Collagenase (Sigma-Aldrich, catalog number: C0130 )
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Trypsin inhibitor (Sigma-Aldrich, catalog number: T2011 )
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KCl (Sigma-Aldrich, catalog number: P3911 )
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NaCl (Sigma-Aldrich, catalog number: S7653 )
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MgCl2·6H2O (Sigma-Aldrich, catalog number: M2670 )
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HEPES (Sigma-Aldrich, catalog number: PHR1428 )
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CaCl2 (Sigma-Aldrich, catalog number: C1016 )
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NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
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MgSO4·7H2O (Sigma-Aldrich, catalog number: M2773 )
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MES (Sigma-Aldrich, catalog number: M3671 )
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Streptomycin (Sigma-Aldrich, catalog number: 5711 )
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Ampicillin (Sigma-Aldrich, catalog number: A5345 )
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Gentamicin sulfate (Sigma-Aldrich, catalog number: G1914 )
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Inorganic salt stock solution (see Recipes)
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Antibiotic stock solutions (see Recipes)
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LB medium (see Recipes)
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LB agar (see Recipes)
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ND96 solution (Ca2+-free) (see Recipes)
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ND96 solution (see Recipes)
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MBS solution (see Recipes)
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K+-Free MBS solution (see Recipes)
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Oocytes isolation solution (see Recipes)
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Extract raw data of the values of K+ content as a text from 4100-MP (Table 1).
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Open the text containing the raw data in Microsoft EXCEL.
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Calculate the K+ efflux of each oocyte by the following formula:
C/N, unit: nmol/oocyte. C represents the measured values, the unit is μmol/L; N represents the number of oocytes in each sample. Average the K+ efflux of quintuplicate with standard deviation.
Table 1. The measured K+ concentration in solution for K+ efflux ability measurement
H2O and LKS2, which are negative controls, represent water-injected and mutated-NRT1.5-injected oocytes, respectively.
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Calculate the K+ amount in each oocyte by the following formula:
CxR/5, unit: nmol/oocyte. C represents the measured values, the unit is μmol/L; N represents the number of oocytes in each sample; R represents dilution rate.
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Average the K+ amount of quintuplicate with standard deviation (Table 2).
Table 2. The measured K+ content in oocytes before and after K+ efflux
H2O and LKS2, which are negative controls, represent water-injected and mutated-NRT1.5-injected oocytes, respectively.
I thank Xiaorong Fan (Nanjing Agricultural University) for providing us with the pT7TS vector. I also thank Yi Wang (China Agricultural University) for discussions and help. This work is supported by the National Natural Science Foundation of China (31800198), the Natural Science Foundation of Jiangsu Higher Education Institutions (18KJB180010), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The adapted protocol for oocytes isolation and injection described in that manuscript has used by Dascal (1987).
There are no conflicts of interest or competing interest.