===== General information for researchers submitting DNA/RNA samples to the Southern California Genotyping Consortium ===== July 2009 Version 8 ==== Genomic DNA samples submitted to the SCGC for GoldenGate genotyping should be prepared according to the following guidelines: ==== Each reaction (1 reaction per oligo pool per sample) requires a minimum of 1 Ug of genomic DNA (250 ng for the reaction plus dead volume and quant check volume) . Liquid DNA samples should be normalized in water or 1 X TE to a standard concentration between 100 ng and 200 ng per ul. Samples with concentrations below 50 ng per ul will not be accepted for genotyping and will be returned to you to be concentrated or re-aliquoted. Please err on the high side if possible. The upper limit is fairly flexible, we ask only that samples be completely resuspended and not at all viscous. Please note that we have found Nanodrop spec concentrations to be somewhat variable. If using a nanodrop or other method that requires pipetting very small volumes (1-2 ul) please consider that you may be significantly overestimating the true concentration. As noted above, in addition to the amount of DNA consumed in the reaction itself (assumed to be 5ul even though the sample may have to be diluted prior to genotyping), all samples must be delivered with an extra 5 ul of DNA per sample to confirm the DNA concentration using a PicoGreen assay plus 10 ul of DNA per sample as dead pipetting volume. Any and all DNA left over at the end of a project can be returned to you if you choose but these extra volumes are not optional. As an example: A single OPA project (96 to 1536 snps) with DNA normalized to 100 ng/ul would require 5ul for the Pico green quant check and 10 ul as pipetting dead volume and 5ul for the reaction itself for 20 ul total volume. If contemplating using Whole Genome Amplification products as samples, please inquire. We require that DNA samples be sent in 96 well format PCR type plates of this specific type: * 96 well skirted v-bottom polypropylene microplates Cat# MSP-9601 from Biorad sealed with strip caps. We’re happy to ship these plates to you if you are unable to obtain the correct plates. In order to avoid cross contamination during transport, it’s best to ship the samples frozen, however, if shipping from outside the US there is a strong possibility that the samples will be delayed by US customs and/or the USDA. You might consider shipping the samples at RT in this case as the dry ice will not last through the inspection process. You should also include a very thorough description of the materials to avoid any unnecessary delay. ** Samples sent in plates other than those listed above will be re-plated into the correct plates and will incur a charge of $100 per plate. ** ==== Genomic DNA samples submitted to the SCGC for Infinium Whole Genome Genotyping should be prepared according to the following guidelines: ==== The amount of DNA required varies slightly according to the specific chip used, however, a standard 15 ul at 100 ng per ul will work for all chips and is the strongly preferred option. If, however, you’d like to reduce the amount of DNA to the minimum required for a specific chip please use the following guidelines when preparing your samples. Please note that we will verify concentrations using a pico green assay. It’s strongly recommended that any samples submitted at 50 ng per ul be quanted with pico green before submission to avoid significant discrepancies. |Duo format chips (1M) | 15 ul @ 50 ng per ul| |Quad format chips (Omni-1/610/660W/CNV370) | 12 ul @ 50 ng per ul| |12 x 1 format chips ( CytoSnp-12) | 12 ul @ 50 ng per ul| |Methylation chips (meth-27) | 23 ul @ 50 ng per ul| In general, liquid DNA samples should be normalized in 1 X TE to a standard concentration between 100 ng and 200 ng per ul, Samples with concentrations below 50 ng per ul will not be accepted for genotyping and will be returned to you to be concentrated or re-aliquoted. Please err on the high side if possible. The upper limit is fairly flexible, we ask only that samples be completely resuspended and not at all viscous. IF SUBMITTING FEWER THAN 96 SAMPLES PER PLATE PLEASE ARRANGE THEM IN COLUMNS RATHER THAN IN ROWS. Please note that we have found Nanodrop spec concentrations to be somewhat variable. If using a nanodrop or other method that requires pipetting very small volumes (1-2 ul) please consider that you may be significantly overestimating the true concentration. Any and all DNA left over at the end of a project can be returned to you if you choose. We require that DNA samples be sent in 96 well format PCR type plates of this specific type: * 96 well skirted v-bottom polypropylene microplates Cat# MSP-9601 from Biorad sealed with strip caps or heat sealed (please use caps if shipping through the mail, foil seals are fine otherwise). We’re happy to ship these plates to you if you are unable to obtain the correct type. In order to avoid cross contamination during transport, it’s best to ship the samples frozen, however, if shipping from overseas there is a strong possibility that the samples will be delayed by US customs and/or the USDA. You might consider shipping the samples at RT in this case as the dry ice will not last through the inspection process. You should also include a very thorough description of the materials to avoid any unnecessary delay. **Samples sent in plates other than those listed above will be re-plated into the correct plates and will incur a charge of $100 per plate. ** Whole Genome Amplification is __not compatible__ with Infinium Whole Genome Genotyping at this time. ==== Human Genomic DNA samples submitted to the SCGC for Infinium Whole Genome Genotyping should be prepared according to the following guidelines: ==== * Each reaction (1 sample=1 reaction) requires 4ug of genomic DNA. * Liquid DNA samples should be normalized in 1 X TE to a standard concentration between 100 ng and 200 ng per ul, Samples with concentrations below 50 ng per ul will not be accepted for genotyping and will be returned to you to be concentrated or re-aliquoted. Please err on the high side if possible. The upper limit is fairly flexible, we ask only that samples be completely resuspended and not at all viscous. Please note that we have found Nanodrop spec concentrations to be somewhat variable. If using a nanodrop or other method that requires pipetting very small volumes (1-2 ul) please consider that you may be significantly overestimating the true concentration. * In addition to the amount of DNA consumed in the reaction itself (750 ng), we also require an extra 5 ul of DNA per sample to confirm the DNA concentration using a PicoGreen assay plus 10-20 ul of DNA per sample as dead pipetting volume. Any and all DNA left over at the end of a project can be returned to you if you choose. Whole Genome Amplification is not compatible with Infinium Whole Genome Genotyping at this time. ==== RNA samples submitted to the SCGC for Gene Expression analysis on the Human, Mouse or Rat genechips should be prepared according to the following guidelines: ==== Input RNA quality is the most significant factor in overall data quality. Therefore it is highly recommended that all total RNA samples be free of all contaminants (Trizol etc) and be assessed for quality using an Agilent Bioanalyzer.((This service can be arranged with the good people at the UCLA microarray facility. Please note that they are completely separate from the SCGC and we are unable to coordinate these assays for you. They can be contacted via email at:\\ Microarray@mednet.ucla.edu\\ Or by phone:\\ Ph: 310-267-1947)) We require 200 ng total RNA in a volume less than 12 ul for each sample plus 5ul additional RNA for a RiboGreen quant check. We confirm the concentration of all samples using a ribogreen assay. Other methods can give results that are quite different than those generated by ribogreen. For the purposes of this assay, we recommend that you assume that your (non-ribogreen) results are high by a factor of 3 and adjust the amount you send to us accordingly. Nothing personal, it just saves time. Gene expression samples should be in microplates or in multi-channel pipette accessible strip tubes. All plates and or strips must be clearly labeled with your name or other identifying information. ==== General notes: ==== Plates with less than 96 samples ( including any controls) should be plated in columns ( A1, B1, C1 etc.) rather than in rows ( A1, A2, A3 etc) to avoid replating. Please note that all submissions must be accompanied by an electronic sample sheet ([[downloads|Available on the SCGC downloads page]]). If you wish for the SCGC to perform Mendel checks on the genotyping results please also provide a family file in linkage format. Please notify me by email when samples have been shipped and include the tracking number (and shipper) so I can keep an eye on their progress My email address is: My phone number is: 1 310 825-2390 The shipping address is:\\ UCLA Gonda building\\ 695 Charles Young Dr. South\\ Los Angeles, California\\ 90095\\ Attn: Joe DeYoung\\ Room 3554\\