Young leaves were collected, frozen in liquid nitrogen, and then homogenized in a Shake Master shaker (Bio Medical Science, Tokyo, Japan). Genomic DNA was isolated using the Genomic-tip 20/G and Genomic DNA Buffer Set (Qiagen GmbH) as described by Yamamoto et al. (2006) . The PCR mixture (10 μL) contained 5 μL of GoTaq Colorless Master Mix (Promega, Madison, WI, USA), 10 pmol each of forward and reverse primers, and 5 ng of genomic DNA. PCR was performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) using the following program: an initial denaturing step at 94°C for 2 min; 35 cycles of denaturation at 94°C for 1 min, annealing at either 52°C, 55°C, 58°C, or 61°C for 1 min, and extension at 72°C for 1 min; and a final extension step at 72°C for 3 min. PCR products were separated in 2% (w/v) agarose gel in TAE buffer and stained with 0.5 μg/mL ethidium bromide or SYBR green I (TaKaRa bio, Shiga, Japan). Amplified fragment sizes were estimated against a 100-bp DNA Ladder (Toyobo, Osaka, Japan).
Genomic tip 20 g and genomic dna buffer set
Manufactured by Qiagen
The Genomic-tip 20/G is a high-capacity anion-exchange resin-based column designed for the purification of genomic DNA. The Genomic DNA Buffer Set provides the necessary buffers for the purification process. These products facilitate the isolation of high-molecular-weight genomic DNA from a variety of sample sources.
Automatically generated - may contain errors
Lab products found in correlation
100 bp dna ladder, by Toyobo (1 mentions)
Sybr green 1, by Takara Bio (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Gotaq colorless master mix, by Promega (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
2 protocols using genomic tip 20 g and genomic dna buffer set
1
Genomic DNA Extraction and PCR Amplification
2
Transcriptomic and Epigenetic Profiling of Manduca sexta
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Samples were taken from our in-house M. sexta stock, which was reared as previously described (Gegner et al., 2019) (link). We selected three female L5 during their feeding stage (2-3 days after molting) and three female adults 2-3 days after their eclosion. Individual insects were frozen and ground in liquid nitrogen. RNA was isolated from 50 mg of ground tissue of one animal using the Direct-zol RNA MiniPrep Kit (ZymoResearch) including DNA digestion according to the manufacturer's recommendations. Sample quantity and purity were assessed using a NanoDrop ND-1000 UV/Vis spectrophotometer (Thermo Fisher Scientific) and the integrity was verified using an Agilent 2100 Bioanalyzer and a RNA 6000 Nano Kit (Agilent Technologies). DNA was extracted from equal amounts of tissue from the same individual using the Genomictip 20/G and Genomic DNA Buffer Set (Qiagen) according to the manufacturer's protocol. RNA-Seq using poly(A)+ enriched RNA fragmented to an average of 250 bp and bisulfite sequencing were carried out using the llumina 2000 HiSeq2500 platform at GATC Biotech (Eurofins Genomics) based on single samples of 2 µg RNA or 1 µg DNA, respectively. Sample preparation for Bisulfite and RNA sequencing was performed by GATC Biotech using their in house protocols.
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