Dl2000 plus dna marker

293T cells were seeded in 12-well plates and grown in 1 ml Dulbecco’s Modified Eagle’s Medium (10% fetal bovine serum). After the cells were 50–70% confluent, 293T cells were transfected with 1 μg of DNA (containing either wild-type or mutant Minigene ATP7B_ex) using Lipofectamine 2000 (Invitrogen, America). At 24 h after transient transfection, total RNA was extracted from cells using TRIzol (Takara, Japan). The first cDNA strand was reversely transcribed using the Superscript III Reverse Transcriptase (Invitrogen, America) together with random hexanucleotide primers. Then, 2 μl of each cDNA was amplified by TaKaRa Ex Taq (Takara, Japan), with primers located in the two cassette exons of the pSPL3 vector. The primer sequences were as follows: forward SD 5′-TCTGAGTCACCTGGACA ACC-3′ and reverse SA 5′-ATCTCAGTGGTATTTGTGAGC-3′. The PCR products were analyzed by agarose gel electrophoresis and proven by sequencing of the extracted DNA. Gel containing 1.5% agarose in Tris-acetate-EDTA buffer was prepared and applied. The marker used in the agarose gels was DL2000 Plus DNA Marker (Vazyme, China). The ratio of normal and abnormal transcripts was analyzed by grayscale value, and the experiment was repeated four times in each group. The mean value was obtained.