Abi veriti pcr thermocycle instrument

Site-directed mutagenesis was performed according to a previous study (23 (link)). Firstly, PCR with pGL3-TCTN1 (−550/-491) as the template was performed, by using a Q5 high-fidelity DNA polymerase (#E0555L, New England Biolabs, Inc.) in an ABI Veriti PCR thermocycle instrument (Applied Biosystems) according to the following protocol: 95°C, 1 min; 95°C, 15 sec; 50°C, 30 sec; 72°C, 5 min for 30 cycles; 72°C, 10 min. The primers used for mutating the TFAP2A-binding site are as follows: Forward, 5′-ACTGCACTCCAATTGTTTATACAGAGTGAG-3′ and reverse, 5′-CTCACTCTGTATAAACAATTGGAGTGCAGT-3′. The products were digested with DpnI enzyme (New England Biolabs, Inc.) and then transferred into the top 10 bacterial E. coli cells (Biomed) for amplification. Mutations were confirmed by Sanger sequencing.

The pGL3-TCTN1 plasmid was used as the template. The primer sequences were designed alongside the deleted region and are listed in the Table SIII. The PCR cycling was performed on an ABI Veriti PCR thermocycle instrument (Applied Biosystems) with the conditions as follows: 95°C, 1 min; 95°C, 15 sec; 55°C, 30 sec; 72°C, 5 min for 30 cycles; 72°C, 10 min. Subsequently, the product was extracted using an Agarose Gel Extraction kit (Biomed, http://www.biomed168.com/). Following incubation with T4 polynucleotide kinase (New England Biolabs, Inc.) at 37°C for 1 h, the product was self-linked using a T4 DNA ligase (New England Biolabs, Inc.) at 37°C for 1 h and transferred into the top 10 competent bacterial E. coli cells (Biomed) for amplification. TCTN1 promoter deletion mutants were confirmed by Sanger sequencing in an Illumina NextSeq 500 instrument (Illumina, CA, USA) at Sangon Biotech Co., Ltd. The primer used for DNA sequencing was: 5′-CTAGCAAAATAGGCTGTCCC-3′.