Dna polymerase

Total RNAs of MEF-WT, Bcl2^−/−MEF, Hela-WT and Nur77^−/−HeLa cells were prepared using Trizol Reagent (Takara Biomedical Technology) according to the Manufacturer's Instructions. The cDNAs were obtained by using a Reverse Transcription Kit (Yeasen Biotechnology). Primers used for PCR amplification using DNA Polymerase (Vazyme Biotech) were based on the DNA sequences of Bcl-2 and Nur77 from the PubMed Database:
Nur77 (F: 5′- ATGCCCTGTATCCAAGCCCAA-3′, S: 5′-TCAGAAGGGCAGCGTGTCCAT-3′);
Bcl-2 (F: 5′-ATGGCGCAAGCCGGGAGAACA-3′, S: 5′- TCACTTGTGGCCCAGGTATGC-3′),
and the amplified products were identified by Agarose Gel Electrophoresis (Biosharp).

The pEGFP-N1 (cat. no. 6085-1; Addgene, Inc.) plasmid was maintained within our laboratory. Normal Donkey serum (cat. no. D9663) was purchased from Sigma-Aldrich; Merck KGaA. Opti-MEM, DMEM high glucose medium, trypsin and FBS were purchased from Gibco; Thermo Fisher Scientific, Inc., and Lipofectamine^® 2000 and TRIzol^® were purchased from Invitrogen; Thermo Fisher Scientific, Inc. Anti-α-tubulin and anti-tyrosinated tubulin antibodies were supplied by Sigma-Aldrich; Merck KGaA. The homologous recombination kit (cat. no. C122-02; Vazyme Biotech Co., Ltd.), DNA Polymerase (cat. no. P508-d1; Vazyme Biotech Co., Ltd.) and PrimeScript™ IV 1st strand cDNA synthesis mix (cat. no. 6215A; Takara Biotechnology Co., Ltd.) were used for construction of the recombinant plasmid, and the water used in the present study was ultra-pure (R≥18 M Ω/cm).

Fresh mouse tail clips were dissolved in tail lysis buffer at 55°C for 12 hours. Genomic DNA was extracted using absolute ethanol and 70% ethanol. Genotyping was performed by polymerase chain reaction using DNA polymerase (Vazyme, Nanjing, China, Cat# P222). To verify the deletion of Slc30a3 in the retina of ACs or RGCs, genomic DNA was extracted and amplified using the primers GPS00001161-Slc30a3-5wt-tF1 and GPS00001161-Slc30a3-5wt-tR1 (primer information is presented in Additional Table 1). The amplicon size for Slc30a3 wild-type (WT) was 202 bp and Slc30a3 was 304 bp (Additional Table 1). The presence of VGAT-Cre was verified using 12785 and oIMR8292 primers (amplicon size 200 bp), and primers 12786 and 12785 were used to identify the WT allele (323 bp) (Additional Table 1). We used C57BL/6J (wt/wt) mice, VGAT^CreZnT3^fl/fl (mut/wt; Slc30a3^fl/fl) mice, and blank control (water) for grouping. The presence of VGLUT2-Cre was determined by polymerase chain reaction using primers 13007 and 32667, with amplicon sizes of 124 (mutant) and 245 (WT) bp (Additional Table 1). We used C57BL/6J (wt/wt) mice, VGLUT2^CreZnT3^fl/fl (mut/wt; Slc30a3^fl/fl) mice, and blank control (water) for grouping.