--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left date: 2015-10-30 00:48:23+00:00 layout: post slug: dna-isolation-geoduck-olympia-oyster-2 title: DNA Isolation – Geoduck & Olympia Oyster categories: - 2015 - Geoduck Genome Sequencing - Olympia Oyster Genome Sequencing tags: - DNA Isolation - DNA Quantification - gDNA - geoduck - NanoDrop1000 - olympia oyster - Ostrea lurida - Panopea generosa --- Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck adductor muscle sample from Box 1 of the geoduck [samples collected by Brent & Steven on 20150811.](https://onsnetwork.org/halfshell/2015/08/11/big-day-big-clam/) Used Olympia oyster ctenidia from Box 1 of [adductor muscle sample collected by Brent & Steven on 20150812.](http://onsnetwork.org/halfshell/2015/08/12/another-day-another-species/) Tissues were split in approximately half, minced and transferred to tubes with 1mL of DNAzol + 50μg/mL of Proteinase K (Fermentas). Previously, I had just homogenized samples. I'm hoping that the overnight digestion with Proteinase K will help increase yields from these. Tissue weights: * Geoduck adductor muscle tube 1: 292mg (gone) * Geoduck adductor muscle tube 2: 320mg (gone) * Olympia oyster ctenidia tube 1: 135mg (gone) * Olympia oyster ctenidia tube 2: 130mg (gone) Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments: * Samples were incubated O/N @ RT on a rotator. * After Proteinase K digestion, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins. * Performed optional centrifugation step (10,000g, 10mins @ RT) * Initial pellet wash was performed using a 70%/30% DNAzol/EtOH * Pellets were resuspended Buffer EB (Qiagen) Resuspension volume = 500μL total for each species Samples were incubated O/N at RT to facilitate pellet resuspension. NOTE: Geoduck "pellets" were not very DNA pellet-like. Very loose, white, and sort of disintegrate (but not dissolve in solution) when attempted to resuspend.