Pcr digoxigenin probe synthesis kit

To investigate the ChPAP transcript levels under high salinity and drought stresses, the TSA Chlorella samples were grown on medium supplemented independently with 200-mm NaCl and 300-mm sorbitol. The samples were collected at 0, 3, 6, 12, 24, and 48h and then ground with a mortar and pestle in liquid nitrogen for RNA isolation.
The total RNA of TSA Chlorella was extracted using RNAiso Plus reagent (TaKaRa, Japan). The ChPAP-specific forward (5'-ATGGGCCTCAAGGAAGAC-3') and reverse (5'-TCAAGCGTACTTCGCCTTCAG-3') primers were used to amplify the cDNA probes using a PCR Digoxigenin Probe Synthesis kit (Roche, Switzerland). The northern blot analysis was performed in accordance with a previously published protocol (Qiao et al., 2018 (link)).

To check the expression level of ChACBP under various abiotic stresses including high salinity, oxidation, and heavy metal stresses, Chlorella cells were grown in liquid medium supplemented with 200 mM NaHCO₃, 200 mM NaCl, 2 mM H₂O₂, or 100 μM CuCl₂, respectively. Low temperature stress was applied by incubating the microalga at 4°C. Samples were collected at 0, 3, 6, 12, 24, and 48 h for analysis. The concentration gradients in the stress treatments were as follows: 0, 50, 100, 200, 300, and 400 mM NaHCO₃; 0, 50, 100, 150, 200, and 300 mM NaCl; 0, 1, 2, 3, 4, and 5 mM H₂O₂; 0, 50, 100, 200, 300, and 500 μM CuCl₂; and temperatures of 24°C, 18°C, 16°C, 14°C, 10°C, and 4°C. Samples were collected at 6 h of these treatments, and ground in liquid nitrogen with a mortar and pestle.
Total RNA was extracted using RNAiso plus (TaKaRa, Kyoto, Japan). Northern blot analysis was performed using the Digoxigenin Nucleic Acid Detection kit (Roche, Basel, Switzerland). Total RNA (3 μg) was separated on a 1.5% agarose gel and transferred to a Hybond-N membrane (Amersham). The ChACBP cDNA probes were obtained using the PCR Digoxigenin Probe Synthesis kit according to the manufacturer’s instructions (Roche). Hybridization was performed according to standard procedures recommended by the manufacturer (Roche), and then detected using the CDP Star system with the LAS-4000 imager.

Genomic DNA was extracted from tail of the transgenic/control mouse by using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China). PCR assay was performed by using the primers shown in Table 1. The 1226 bp PCR product contains partial K14 promoter and partial ovine β-catenin DNA sequence. The PCR procedure: 5 min at 95°C, 35 cycles of 30 s for 95°C, 30 s for 58°C and 72°C for 1 min, 72°C for 10 min and hold at 4°C forever. The further southern blot assay, digestion: the BstEⅡ was selected for digested the DNA (60°C, 20 h), which is obtained from mouse tail. The probe (739 bp) amplified by primer (F6 and R6 in Table 1) across promoter and β-catenin sequence, was labeled with the PCR digoxigenin probe synthesis kit (Roche). The DIG-High Prime DNA Labeling and Detection Starter KitⅡ was used for washing and hybridization (Roche, Basel, Switzerland).