DNA Extraction and PCR Amplification

Genomic DNA was extracted from the fin clips with the DNeasy 96 Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. PCR was carried out using Colorless GoTaq Flexi Reaction Buffer (Promega) with PCR dnd primers^{19 (link)}. The PCR product was purified with QIAquick PCR purification Kit (Qiagen) and measured via Qubit Fluorometric Quantitation (Invitrogen). Purified and quantified PCR products were cloned into PCR4-TOPO using the TOPO TA cloning kit for sequencing (Invitrogen). Cloned fragments were sequenced with M13 forward and reversed primers using BigDye 3.1.

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Güralp H., Skaftnesmo K.O., Kjærner-Semb E., Straume A.H., Kleppe L., Schulz R.W., Edvardsen R.B, & Wargelius A. (2020). Rescue of germ cells in dnd crispant embryos opens the possibility to produce inherited sterility in Atlantic salmon. Scientific Reports, 10, 18042.

Publication 2020

DNeasy 96 Blood and Tissue Kit Colorless GoTaq Flexi Reaction Buffer QIAquick PCR purification Kit Qubit Fluorometric Quantitation TOPO TA cloning kit for sequencing

Blood Buffer Clips Fluorometric Genomic Primers Promega Tissue Topo

Corresponding Organization :

Other organizations : Norwegian Institute of Marine Research

Top 5 similar protocols

Variable analysis

independent variables

PCR primers
Cloning into PCR4-TOPO vector

dependent variables

PCR product sequence

control variables

Genomic DNA extraction method (DNeasy 96 Blood and Tissue Kit)
PCR reaction buffer (Colorless GoTaq Flexi Reaction Buffer)
PCR product purification method (QIAquick PCR purification Kit)
Cloning method (TOPO TA cloning kit for sequencing)
Sequencing method (BigDye 3.1)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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