DNA extraction. The majority of samples that pass pathology review are able to have DNA isolated. A small percentage of samples yield <50 ng of DNA, which is currently the minimum amount of DNA needed for the FMI assay. If this occurs, the Sponsor Contact is contacted to decide if additional sample is available, and, in consultation with FMI decide whether to attempt to continue the assay.
DNA extraction. The Nucleon II kit was used according to the manufacturer’s instructions (see appendix).
DNA extraction. For the new samples for which no DNA was available, DNA was extracted from newly collected samples. DNA was extracted using the Wizard® Genomic DNA Purification kit (Promega, WI, USA) following manufacturer´s instructions for “Isolating Genomic DNA from Tissue Culture Cells and Animal Tissue”. The starting material was approximately 20 mg of tissue and after extraction all samples were suspended in equal volumes of Milli-Q water. DNA quantity (ng/µl) was evaluated on the Qubit® 2.0 Fluorometer (Life Technologies) and DNA integrity was assessed by electrophoresis.
DNA extraction. Well water- DNA was extracted according to reference1. Filters were aseptically removed from casing and cut into pieces, and processed using the UltraCleanTM Water DNA Isolation kit (Mo Bio) according to the manufacturer’s instructions using the alternative lysis method (65 °C incubation for 10 minutes). DNA was stored at −20 °C until analysis. Drill-wetting mud- DNA was extracted using the Omega X.X.X.X.XX Soil DNA Kit. Samples were processed according to manufacturer’s instructions using 300 mg of starting material, vortexing for 5 minutes to lyse samples, and eluting in a final volume of 50 L of elution buffer supplied with the kit. DNA was stored at −20 °C until analysis.
DNA extraction. DNA was extracted from the xxxxx xxxxx using the Illustra blood genomicprep spin kit, (GE Healthcare, UK).
DNA extraction. DNA extraction from xxxxx xxxxx of 466 RISCK subjects and 310 MARINA subjects was carried out using an Illustra blood genomic prep mini spin kit (GE healthcare, UK). Xxxxx xxxxx in EDTA had been frozen since preparation of blood samples. Genomic DNA was purified from 200 μl buffy coat. This process was carried out according to manufacturer’s instructions. This includes the below steps:
1- Blood cell lysis: 20 μl of Proteinase K was added into the bottom of a 1.5 ml microcentrifuge tube. This digests protein, removing contamination and inactivating nucleases that might otherwise degrade the DNA during purification. This was followed by adding 200 μl of the buffy coat and then 400 μl lysis buffer into the same tube. The tube was mixed well for 15 seconds and incubated at room temperature for 10 minutes.
2- Genomic DNA binding: a mini column was assembled in a collection tube. The sample was loaded on to the centre of the column and was centrifuged. The flow through in the collection tube was discarded. The column was placed back inside the collection tube.
3- Wash: to ensure complete cell lysis and to denature any residual protein; 500 μl of lysis buffer was added to the column and centrifuged. And again, flowthrough in the collection tube was discarded.
4- Wash and dry: into the column, 500 μl of wash buffer was added. This was centrifuged for 3 minutes. The collection tube and flow through were discarded.
5- Elution: the purification column was transferred into a fresh DNAse- free microcentrifuge tube. Followed by adding 200 μl of 70 oC preheated elusion buffer on the centre of the column. This was centrifuged for 1 minute. Purified genomic DNA was stored at -20 oC.
DNA extraction. Completed Sample Prep
