Primers were designed to synaptotagmin-1 cDNA (NM_001252342.1) and extended by a BamHI site at the 5′ end and an AsiSI site at the 3′ end (Supplementary Table 1 ). Syt1 was amplified using the Phusion MasterMix PCR protocol (NEB). The vector FUchW H2KK-T2A-Venus, which contains the T2A cleavage peptide sequence, and the Syt1 PCR amplicon were digested with BamHI and AsiSI, gel-purified, ligated with Quick ligation protocol (NEB) and transformed in NEB 10-beta competent cells. Clones were picked, QuickLyse miniprep (Qiagen) performed and the plasmid send for sequencing. The positive clone was retransformed in Stable-3 cells and plasmid purified with PureLink HiPure Plasmid DNA Purification kit (Invitrogen).
Phusion mastermix pcr protocol
Manufactured by New England Biolabs
Phusion MasterMix is a high-fidelity, premixed PCR solution that contains all the necessary reagents for efficient and accurate DNA amplification. It is designed to provide consistent and reliable results with a wide range of DNA templates.
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2 protocols using phusion mastermix pcr protocol
1
Synaptotagmin-1 expression plasmid construction
2
Synaptotagmin-1 expression plasmid construction
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Primers were designed to synaptotagmin-1 cDNA (NM_001252342.1) and extended by a BamHI site at the 5′ end and an AsiSI site at the 3′ end (Supplementary Table 1 ). Syt1 was amplified using the Phusion MasterMix PCR protocol (NEB). The vector FUchW H2KK-T2A-Venus, which contains the T2A cleavage peptide sequence, and the Syt1 PCR amplicon were digested with BamHI and AsiSI, gel-purified, ligated with Quick ligation protocol (NEB) and transformed in NEB 10-beta competent cells. Clones were picked, QuickLyse miniprep (Qiagen) performed and the plasmid send for sequencing. The positive clone was retransformed in Stable-3 cells and plasmid purified with PureLink HiPure Plasmid DNA Purification kit (Invitrogen).
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