Genomic DNA was isolated using the Easy DNA Kit (Invitrogen). Plasmid DNA was extracted with QIAprep spin Miniprep Kit (Qiagen). DNA fragments were amplified using Takara Ex Taq DNA polymerase (Takara) or Pfu Turbo DNA Polymerase (Agilent Technologies). PCR products were isolated and purified using QIAquick PCR gel extraction kit (Qiagen). Custom DNA oligonucleotides (see Table S5 ) were purchased from Sigma-Aldrich. For cloning purposes, restriction endonuclease and T4 DNA ligase were obtained from New England Biolabs Inc. (NEB). The QuikChange site-directed mutagenesis system (Stratagene) was used to introduce in vitro point mutations following the manufacturer’s instructions.
Custom dna oligonucleotides
Manufactured by Merck Group
Custom DNA oligonucleotides are short, synthetic DNA sequences designed to meet specific research requirements. These DNA strands can be customized in length, sequence, and chemical modifications to serve a variety of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Takara ex taq dna polymerase, by Takara Bio (2 mentions)
Easy dna kit, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr gel extraction kit, by Qiagen (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Restriction endonuclease, by New England Biolabs (2 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (1 mentions)
Platinum superfi 2 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
3 protocols using custom dna oligonucleotides
1
Molecular Cloning and Mutagenesis Protocol
2
Cloning and Tagging Killifish Hormone Genes
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Killifish cDNAs were cloned from brain tissues from male and female killifish by homogenization in TRIreagent (Sigma #T9424) using 3 mm Nirosta disruption beads (PALBOREG FEDERAL #BL6693003000) and a tissue homogenizer (TissueLyser LT, Qiagen #85600). Total RNA was isolated from the lysed tissues using the Direct-zol RNA miniprep kit. (Zymo research #R2052), and the Verso cDNA Synthesis Kit (Thermo scientific #AB1453A) was used to prepare cDNA with random primers according to the manufacturer’s protocol. cDNA for the gh1 and fshb was amplified using custom DNA oligonucleotides (Sigma) and Platinum SuperFi II DNA Polymerase (Invitrogen, #12361010). Primer sequences are available in the Key Resources Table . PCR products were purified (QIAquick PCR purification kit, Qiagen #28104) and sequence-verified. The sequence-verified ORFs were cloned using GIBSON (NEB, #E2611L) into the pLV-EGFP plasmid or our Dox inducible plasmid (#196331), which was modified such that each hormone is tagged with a GFP, separated by the T2A self-cleaving peptide (Liu et al., 2017 (link)). Plasmids and corresponding annotated maps are available via Addgene (#194356, #194883, #196331, # 205595, # 205596, #205597).
3
Molecular Cloning and Mutagenesis Protocols
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Genomic DNA was extracted with an Easy DNA kit (Invitrogen). Plasmid DNA was obtained with a QIAprep Spin miniprep kit (Qiagen). PCR products were obtained using Pfu Turbo DNA polymerase (Stratagene) or TaKaRa Ex Taq DNA polymerase (TaKaRa). PCR products were purified from an agarose gel using a QIAquick PCR gel extraction kit (Qiagen). Custom DNA oligonucleotides (see Table S4 in the supplemental material) were purchased from Sigma-Aldrich. Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs Inc. (NEB). The QuikChange site-directed mutagenesis system (Stratagene) was used to introduce in vitro point mutations per the manufacturer’s instructions. Nucleic acid quantification was carried out using NanoDrop 8000 (Thermo Scientific).
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