GENOMIC DNA SAMPLE REQUIREMENTS WHAT WE REQUIRE Samples from each individual to be sequenced must contain at least 3ug of high molecular weight (>50kb) DNA at a concentration >30ng/uL. DNA must be purified, of high quality, and free of non-target contaminant nucleic acid (RNA, plastid/bacterial DNA). DNA must also be normalized to have equal concentrations across each sample. To meet these requirements, please use the following instructions. EXTRACT DNA We recommend using Qiagen’s DNeasy Blood & Tissue and DNeasy Plant kits to isolate genomic DNA. Genomic DNAs prepared using these kits routinely generate high quality RAD libraries and sequence data. DNA sample inputs are critical to the success of the RAD procedure; please consider this if you plan to use a different type of kit or homemade protocol. Feel free to contact the lab staff ([email protected]) prior to DNA isolation with questions regarding extraction methods. QUANTIFY Samples from each individual to be sequenced must contain at least 3ug of high molecular weight (>50kb) DNA at a concentration >30ng/ uL. Because DNA quantification determined using spectrophotometry can vary between labs, overestimate DNA concentration, and is sensitive to DNA purity, we recommend that DNA concentration be assessed by fluorometry. We recommend using the Qubit Quant iT Assay system (Invitrogen). CHECK QUALITY We ask that you visualize your samples on an agarose gel to check for degradation. Additionally, we highly recommend using a semi-quantitative ladder to compare measured concentrations with the gel image to verify accuracy of quantification measurements. NORMALIZE RAD sequencing on two or more samples requires DNA concentration to be normalized (equivalent concentrations) across each sample to be analyzed. Samples must be normalized to a concentration of 30ng/uL or greater. It is highly recommended that you run a gel with normalized material to visually check that samples appear to be of equal concentrations, as samples that do not adhere to this standard will not be processed. SEND SAMPLE INPUT AND QUALITY DOCUMENTATION Send an electronic copy of your gel image(s) and sample manifests to [email protected] for quality approval before shipping your samples. Wait for approval from lab staff before continuing with shipment. PREPARE SAMPLES FOR SHIPMENT • 1-7 samples: Samples should be shipped in 1.5-2mL tubes, each clearly labeled with a sample name and shipment date. Wrap the tops of your tubes in paraﬁlm and place them in a plastic conical or other hard container stuffed with tissue to prevent damage during transport. • >7 samples: Samples should be shipped in 96-well plates, each clearly labeled with a plate name and shipment date. Please seal the plates in plastic bags and pad them with bubble wrap or paper towels to prevent damage during transport. Samples shipped in individual tubes will be returned. www.floragenex.com