Genomic DNA (gDNA) was extracted from approximately 600 mg of Stevia leaves using cetyltrimethylammonium bromide (CTAB)-based extraction method [45 ]. The final gDNA pellet was washed with ice-cold 75% ethanol and dissolved in water.
PCR amplification was carried out from 100 ng of gDNA extracted from each line of transgenic Stevia to check for the presence of T-DNA using forward primers specific to the CaMV 35S promoter and reverse primers specific to the 3′-end of SrDXS1 or SrKAH (Additional file9 : Table S1).
Southern blot analysis for detection of transgene integrations and number of integration sites was performed using a DIG-labelled probe specific to the full-length nptII (Roche). The purity of the synthesized probes was checked by electrophoresis on a 1% agarose gel. gDNAs extracted from the SrDXS1-OE and SrKAH-OE lines were digested with HindIII and XbaI, respectively. After digestion, the fragments were resolved on a 0.8% agarose gel together with DIG-labelled DNA molecular weight marker II (Roche). The agarose gel was treated with 0.2 M HCl followed by denaturation solution (0.5 M NaOH, 1.5 M NaCl) and neutralization solution (1 M Tris-Cl pH 7.4, 1.5 M NaCl) and transferred to a positively charged nylon membrane (Hybond-N+, GE healthcare life sciences) in 20x SSC (3.0 M NaCl, 0.3 M sodium citrate, pH 7.0). After the transfer, UV-crosslinking was carried out using Stratalinker 2400 (Stratagene, USA). Then, DIG-based Southern blot hybridization was performed according to manufacturer’s instructions (Roche). Chemiluminescence from the membrane was acquired with the ChemiDoc Touch Imaging System (Bio-Rad, USA).
PCR amplification was carried out from 100 ng of gDNA extracted from each line of transgenic Stevia to check for the presence of T-DNA using forward primers specific to the CaMV 35S promoter and reverse primers specific to the 3′-end of SrDXS1 or SrKAH (Additional file
Southern blot analysis for detection of transgene integrations and number of integration sites was performed using a DIG-labelled probe specific to the full-length nptII (Roche). The purity of the synthesized probes was checked by electrophoresis on a 1% agarose gel. gDNAs extracted from the SrDXS1-OE and SrKAH-OE lines were digested with HindIII and XbaI, respectively. After digestion, the fragments were resolved on a 0.8% agarose gel together with DIG-labelled DNA molecular weight marker II (Roche). The agarose gel was treated with 0.2 M HCl followed by denaturation solution (0.5 M NaOH, 1.5 M NaCl) and neutralization solution (1 M Tris-Cl pH 7.4, 1.5 M NaCl) and transferred to a positively charged nylon membrane (Hybond-N+, GE healthcare life sciences) in 20x SSC (3.0 M NaCl, 0.3 M sodium citrate, pH 7.0). After the transfer, UV-crosslinking was carried out using Stratalinker 2400 (Stratagene, USA). Then, DIG-based Southern blot hybridization was performed according to manufacturer’s instructions (Roche). Chemiluminescence from the membrane was acquired with the ChemiDoc Touch Imaging System (Bio-Rad, USA).
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