High-molecular-weight (HMW) gDNA was extracted from the legs of flash-frozen spiders using Genomic-tips 20/G (Qiagen) based on previous studies [9 (link)]. The specimens were gently and quickly homogenized using a BioMasher II homogenizer (Funakoshi) and mixed with 2 ml of Buffer G2 (Qiagen), including 200 µg ml−1 RNase A and 50 µl proteinase K (20 mg ml−1). After incubation at 50°C for 12 h on a shaker (300 r.p.m.), the mixed lysate was centrifuged at 5000g for 5 min at 4°C, and the aqueous phase was loaded onto a pre-equilibrated QIAGEN Genomic-tip 20/G by gravity flow and washed three times. The DNA was eluted with a high-salt buffer (Buffer QF) (Qiagen), desalted and concentrated using isopropanol precipitation and resuspended in 10 mM Tris–HCl (pH 8.5). The extracted gDNA was qualified using a TapeStation 2200 instrument with genomic DNA Screen Tape (Agilent Technologies) and quantified using a Qubit Broad Range dsDNA assay (Life Technologies). The purified gDNA was size-selected (greater than 10 kb) with a BluePippin with High Pass Plus Gel Cassette (Sage Science).
Biomasher 2
Manufactured by Funakoshi
The BioMasher II is a laboratory tissue homogenizer designed for efficient sample preparation. It is capable of homogenizing a wide range of biological samples, including tissues, cells, and other materials, in order to extract and isolate their components for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Genomic tip 20 g, by Qiagen (7 mentions)
Buffer g2, by Qiagen (4 mentions)
Buffer qf, by Qiagen (4 mentions)
Tapestation 2200, by Agilent Technologies (4 mentions)
Genomic dna screentape, by Agilent Technologies (4 mentions)
Qubit dsdna broad range assay, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Genomic tips 20 g, by Qiagen (1 mentions)
Pvdf blocking reagent for can get signal, by Toyobo (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
5 protocols using biomasher 2
1
High-Molecular-Weight Spider gDNA Extraction
2
Extracting Genomic DNA from Juvenile E. variegata
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Genomic DNA (gDNA) was extracted from the juvenile E. variegata whole body using Genomic-tip 20/G (QIAGEN) following the manufacturer’s protocol. To keep the high molecular weight (HMW) quality, every step was performed as gently as possible. gDNA was extracted from about half of the frozen body. The specimen was homogenized using BioMasher II (Funakoshi) and mixed with 2 ml of Buffer G2 (QIAGEN) including 200 µg/ml RNase A. After the addition of 50 µL Proteinase K (20 mg/mL), the lysate was incubated at 50 °C for up to 12 h on a shaker (300 r.p.m.). The lysate was centrifuged at 5,000×g for 5 min at 4 °C to pellet the debris, and the aqueous phase was loaded onto a pre-equilibrated QIAGEN Genomic-tip 20/G (QIAGEN) by gravity flow. The QIAGEN Genomic-tip 20/G (QIAGEN) was then washed three times, and the DNA was eluted with a high-salt buffer (Buffer QF) (QIAGEN). The eluted DNA was desalted and concentrated by isopropanol precipitation and resuspended in 10 mM Tris-HCl (pH 8.0). Extracted gDNA was quantified using a Qubit Broad Range (BR) dsDNA assay (Life Technologies) and qualified using TapeStation 2200 with genomic DNA Screen Tape (Agilent Technologies).
3
Extracting High-Molecular-Weight Spider gDNA
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According to a previous report48 (link), HMW gDNA was extracted from the whole bodies of flash frozen spiders using Genomic-tip 20/G (QIAGEN). The specimens were quickly homogenized using BioMasher II (Funakoshi) and mixed with 2 mL of Buffer G2 (QIAGEN), including 200 µg/mL RNase A. After the addition of 50 µL of proteinase K (20 mg/mL), the lysate was incubated at 50 °C for 12 h on a shaker (300 rpm). The lysate was centrifuged at 5,000 × g for 5 min at 4 °C to pellet the debris, and the aqueous phase was loaded onto a pre-equilibrated QIAGEN Genomic-tip 20/G (QIAGEN) by gravity flow. The QIAGEN Genomic-tip 20/G (QIAGEN) was then washed three times, and the DNA was eluted with high-salt buffer (Buffer QF) (QIAGEN). The eluted DNA was desalted and concentrated by isopropanol precipitation and resuspended in 10 mM Tris–HCl (pH 8.5). Extracted gDNA was qualified using a NanoDrop 2000 (Thermo Scientific) and TapeStation 2200 with genomic DNA Screen Tape (Agilent Technologies) and quantified using a Qubit Broad Range (BR) dsDNA assay (Life Technologies).
4
Spider Genomic DNA Extraction Protocol
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gDNA was extracted from four adult A. ventricosus whole bodies using Genomic-tip 20/G (QIAGEN) basically following the manufacturer’s protocol. To keep the HMW quality, every step was performed as gently as possible. Flash frozen spider specimens were separated into each body segment, and gDNA was extracted from the cephalothorax and legs. The specimens with the abdomen removed were homogenised using BioMasher II (Funakoshi) and mixed with 2 ml of Buffer G2 (QIAGEN), including 200 µg/ml RNase A. After addition of 50 µL Proteinase K (20 mg/mL), the lysate was incubated at 50 °C for up to 12 h on a shaker (300 rpm). The lysate was centrifuged at 5,000 × g for 5 min at 4 °C to pellet the debris, and the aqueous phase was loaded onto a pre-equilibrated QIAGEN Genomic-tip 20/G (QIAGEN) by gravity flow. The QIAGEN Genomic-tip 20/G (QIAGEN) was then washed three times and the DNA was eluted with high-salt buffer (Buffer QF) (QIAGEN). The eluted DNA was desalted and concentrated by isopropanol precipitation and resuspended in 10 mM Tris-HCl (pH 8.5). Extracted gDNA was quantified using a Qubit Broad Range (BR) dsDNA assay (Life Technologies) and qualified using TapeStation 2200 with genomic DNA Screen Tape (Agilent Technologies).
5
Western Blot Analysis of Fly Heads
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Five heads of 3-day-old female adult flies were homogenized in 150 μl of sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 4% SDS, 0.01% bromophenol blue dye, 10% 2-β-mercaptoethanol) with Biomasher II (Funakoshi). Homogenized heads were boiled for 5 min and then centrifuged at 10,000g for 3 min at 25 °C. Ten microliter of each supernatant was loaded on 5 to 20% gradient polyacrylamide gels (Atto) and then transferred onto Immun-Blot PVDF membranes (Bio-Rad). Membranes were blocked with PVDF blocking reagent for Can Get Signal (TOYOBO) at RT for 1 h and then incubated overnight at 4 °C with diluted primary antibodies. After washing three times with TBST, membranes were incubated at RT for 1 h with HRP-labeled secondary antibodies, then washed three times with TBST again. The chemiluminescent signals were visualized using ImmunoStar Zeta or ImmunoStar LD (Wako) and captured by Amersham Imager 600 (GE healthcare). Signals were quantified with ImageJ software (NIH).
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