Diploid colonies or spore colonies from tetrads were independently cultured overnight at 30° in YPD liquid medium. Genomic DNA was extracted from each culture using the PrepEase DNA isolation kit from Affymetrix following the manufacturer’s protocol. Whole genome sequencing was performed on Illumina HiSequation 2500 machines at Fasteris SA, Switzerland.
Prepease dna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PrepEase DNA isolation kit is a laboratory product designed for the extraction and purification of DNA from various biological samples. The kit provides the necessary reagents and protocols to efficiently isolate high-quality DNA for downstream applications, such as PCR, sequencing, or further analysis.
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5 protocols using prepease dna isolation kit
1
Whole Genome Sequencing of Yeast
2
Isolation and Characterization of Schistosoma japonicum Eggs
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Genomic DNA from S. japonicum was extracted from eggs using the PrepEase DNA isolation kit (Affymetrix, Santa Clara, USA) according to the manufacturer’s guidelines. DNA concentration was quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, USA) and used as positive control.
Eggs of S. japonicum were isolated from the liver of infected mice as previously described [23 (link)]. An aliquot of 50 µl from the resultant isolated egg suspension was then added to 50 µl of lysis buffer and then cracked using a handheld mortar and pestle (Kimble, Vineland, NJ, USA) as previously described [20 (link)]. The homogenised material was then kept at − 20 °C until required for use as a positive control in the LAMP experiments.
When used for the positive control samples, the DNA dipstick was dipped into the homogenised eggs samples five times, dipped in wash buffer five times and into the LAMP reaction tube 15–20 times.
Eggs of S. japonicum were isolated from the liver of infected mice as previously described [23 (link)]. An aliquot of 50 µl from the resultant isolated egg suspension was then added to 50 µl of lysis buffer and then cracked using a handheld mortar and pestle (Kimble, Vineland, NJ, USA) as previously described [20 (link)]. The homogenised material was then kept at − 20 °C until required for use as a positive control in the LAMP experiments.
When used for the positive control samples, the DNA dipstick was dipped into the homogenised eggs samples five times, dipped in wash buffer five times and into the LAMP reaction tube 15–20 times.
3
Genomic DNA extraction and sequencing
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Spore colonies from tetrads were independently cultured overnight at 30° in 4 ml of YPD liquid medium. DNA was extracted from each culture using the PrepEase DNA isolation kit from Affymetrix following the manufacturer’s protocol. Genomic DNA fragmentation and library preparation were performed as described previously (Wilkening et al. 2013 (link)). Briefly, 2 μg of genomic DNA was sheared using a Bandelin Sonorex RX 102 sonicating water bath to obtain DNA fragments of 250–500 bp. End repair, dA-tailing, and ligation were done as per the Illumina library preparation protocol with heat inactivation instead of column/magnetic bead- based cleanups. The multiplexed libraries were amplified and size selected for 350–-400 bp using Invitrogen E-Gel (SizeSelect 2%). The size-selected DNA was sequenced (100PE) on Illumina HiSeq 2000 machines at the EMBL Genomics Core Facilities (GeneCore), Heidelberg, Germany. The raw reads obtained were demultiplexed using the FASTX-toolkit (https://github.com/agordon/fastx_toolkit ). Demultiplexed reads were processed for quality control (QC) using the NGS QC Toolkit (Patel and Jain 2012 (link)). These QC-filtered high-quality reads were used for further analysis. The sequence data are available from the National Centre for Biotechnology Information Sequence Read Archive under accession no. SRP041856.
4
Genome Sequencing of MA Lines
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The parent strains and colonies from their respective MA lines (100th bottleneck only) were independently cultured overnight at 30° in YPD liquid medium. The DNA was isolated from each culture using the PrepEase DNA isolation kit from Affymetrix following the manufacturer’s protocol. The genomic DNA was sequenced using Illumina Hi- Seq 2500 platform at Fasteris, Switzerland.
5
Whole Genome Sequencing of Yeast Mutants
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Spore colonies from the four spore viable tetrads of mlh3 ∆, pch2 ∆, and mlh3 ∆ pch2 ∆ were grown overnight at 30° in 4 ml YPD broth. DNA was isolated from the culture using PrepEase DNA isolation kit (Affymetrix). Whole genome sequencing on Illumina platform was performed at Fasteris, Switzerland, as well as at the European Molecular Biology Laboratory (EMBL) Genomics Core Facilities (GeneCore), Heidelberg, Germany.
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