GSM5292505: 10PAT_37℃_dup1; Homo sapiens; OTHER
Genomic DNA (gDNA) was purified using Blood Genomic DNA Mini Kit (CW2087M, CWbio). PCR reactions were carried out on gDNA to amplify mutant p53 DNA fragments. Each reaction contained a total volume of 50 μL with 1 μg gDNA, 25 μL of VAHTS HiFi Amplification Mix (N616-01, Vazyme) and 2.5 μL of each (5 μM) primer. The parameters for PCR were 95℃ for 3 min, 27 cycles of 98 ℃ for 20 s, 58 ℃ for 15 s, and 72 ℃ for 45 s, followed by one cycle of 72 ℃ for 5 min. The primers used for this reaction were TCCGCCTCCTCTTCCTCCAT (Forward) and ACACCGGCCTTATTCCAAGC (Reverse). Amplicons were separated from non-specific fragments by electrophoresis on a 1% agarose gel stained with GelSafe (EP106-01, DANAGEN), extracted from the gel, and purified using a Wizard® SV Gel and PCR Clean-Up System (A9282, Promega). Sequencing libraries were constructed from 50 ng of purified amplicons using an TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme).