The new CSDaV-derived vectors as well as the additional plasmids from this work were constructed using the NEBuilder HiFi DNA Assembly Cloning system (NEB, Ipswich, MA, USA), following the manufacturer's instructions. The set of primers (Table S1) used in the construction of each plasmid was designed using the SnapGene software version 3.3 (http://www.snapgene.com/). The vectors and insert fragments were obtained by PCR with their respective primers using CloneAmp HiFi PCR premix (Clontech), following the manufacturer's protocol. The amplicons were gel purified using Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corporation, Irvine, CA, USA) and the HiFi DNA Assembly reactions were performed by combining 100 ng of the vector, 100 ng of the respective fragment and 1x of HiFi DNA Assembly premix. The reactions were incubated at 50 °C for 15 min and then transformed into Escherichia coli (DH5α) competent cells. Plasmids were purified from the transformed colonies using the QIAprep Spin Miniprep Kit (Qiagen, Valentia, CA, USA) and confirmed by Sanger sequencing using primers designed to cover the insert region (Table S1).
Nebuilder hifi dna assembly cloning system
Manufactured by New England Biolabs
Sourced in United States
The NEBuilder HiFi DNA Assembly Cloning system is a tool for seamlessly joining multiple DNA fragments in a single, efficient reaction. It enables the rapid and reliable construction of various DNA constructs, such as plasmids, expression vectors, and other genetic elements.
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2 protocols using nebuilder hifi dna assembly cloning system
1
Plasmid Construction Using HiFi Assembly
2
Calcium Channel Chimera Construction
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The following cDNAs were used: Cav2.1 (NM_001127221 ), Cav2.2 e37a (AF055477 ), Cav2.2 e37b (NM_147141 ), β2A (NM_053851 ), and α2δ-1 (NM_000722 .3). The plasmid for β2a-CaM was a gift from I. Dick (University of Maryland, Baltimore, MD). Chimeras were constructed using NEBuilder HiFi DNA Assembly Cloning System (New England Biolabs) and Cav2.1 and Cav2.2 e37a as templates. The following constructs were generated by swapping the amino acids indicated in parentheses: Cav2.2-CT2.1, Cav2.1-CT2.2 (1,681–2,334 of Cav2.2, 1,786–2,261 of Cav2.1); Cav2.2-EF2.1, Cav2.1-EF2.2 (1,681–1,788 of Cav2.2, 1,786–1,892 of Cav2.1); Cav2.2-pre-IQ-IQ2.1, Cav2.1-pre-IQ-IQ2.2 (1,789–1,875 of Cav2.2, 1,893–1,985 of Cav2.1); and Cav2.2-CBD2.1, Cav2.1-CBD2.2 (1,912–1,990 of Cav2.2, 2,009–2,084 of Cav2.1). Additional chimeric channels containing subsets of the EF-hand, pre-IQ, IQ, and CBD were generated using the residues indicated above. For Cav2.2 Δe46, the sequence encoding exons 42–45 of Cav2.2 (1,927–2,162) followed by a stop codon was amplified by PCR and cloned into the corresponding site of Cav2.2 as an XbaI fragment. All chimeras and Cav2.2 Δe46 constructs were cloned into the pcDNA6V5His vector. For generating glutathione S-transferase (GST) fusion proteins, sequences corresponding to theaforementioned Cav2 domains were amplified by PCR and cloned into BamHI and XhoI sites of the pGEX-4T-1 vector.
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