QIAquick Gel Extraction Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
The amount of agarose excised from the gel should be as small as possible.	Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Upstream tips
The amount of agarose excised from the gel should be as small as possible.
Protocol tips
Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.

Publication protocol

Total RNA was extracted from whole bodies of the No. 1 larvae (n = 5) using ISOGEN (NipponGene, Tokyo, Japan). The extracted RNA was purified by DNase treatment to remove genomic DNA. RNA purity and quantity were measured using a NanoVue spectrophotometer (GE Healthcare BioSciences, Tokyo, Japan). The purified RNA (2 µg) was reverse-transcribed for cDNA synthesis using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). ZnNLaz1 gene-specific primers, to amplify the ORF and 5′ and 3′ UTR regions, were newly designed based on de novo assembled sequences obtained using Primer3Plus [19] (electronic supplementary material, table S1). PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Tokyo, Japan), and subcloned into a pGEM easy T-vector (Promega, Madison, WI, USA). The inserted DNA sequence was amplified by PCR, and purified products were sequenced using a BigDye Terminator v. 3.1 Cycle Sequencing Kit and an automatic DNA Sequencer 3130 Genetic Analyzer (Applied Biosystems). Obtained sequences were subjected to BLAST database searches. The determined nucleotide and putative amino acid sequences are available at DDBJ/EMBL/GenBank databases (accession no. LC382016). NLaz homologues in other insects were obtained from the NCBI database, and molecular phylogenetic analyses were performed (see the electronic supplementary material)

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Manufacturer protocol

Download the product protocol from Qiagen for QIAquick Gel Extraction Kit below.

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