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If your species has been previously optimized for GBS, skip this step. Each new species requires optimization for GBS. Test libraries are made to assess restriction enzymes for suitability with your species and to determine the optimal ratio of adapters to DNA. For optimization of your DNA, please supply us with about 10 µg (measure using an intercalating dye or agarose gel rather than absorbance). We cannot use samples with concentrations less than 50 ng/µL. The DNA may be from a single individual or a composite from several individuals of the same species. Label the tube of DNA with the species name. Your optimization sample may be sent prior to your plates and no upload is required for the optimization sample.
Ship your clearly labeled optimization sample to:
Sharon Mitchell, Ph.D.
Genomic Diversity Facility
Biotechnology Resource Center
Institute of Biotechnology
151 Biotechnology Building
Ithaca, NY 14853-2703
Tel: (+1) 607-254-4849
The success of genotyping by sequencing (GBS) is largely determined by DNA quality (molecular weight and purity) and quantity. Below are the guidelines for submitting DNA samples for high-throughput genotyping. If you cannot meet the specifications and/or have questions or concerns, please contact the lab at email@example.com.
Uncut DNA example
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Restriction-enzyme-cut DNA example
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To evaluate the quality of your DNA samples, we require submission of well-labeled gel image(s) containing an aliquot of each DNA prep. Run 100ng of each DNA sample on 1% agarose gels along with 500ng of two λ HindIII size/mass standards per gel. Run the gel long enough so that the size standard bands are clearly separated.
We also require an image of trial restriction enzyme digestions (10% of samples from each plate). Digest 300-500 ng of selected sample DNAs with a restriction enzyme that is NOT methylation sensitive (i.e., HindIII or EcoRI) and run on 1% agarose gels along with the λ HindIII size standards. Use 1 Unit of restriction enzyme and digest for 2 hours at 37 degrees C. These gel images allow us to judge the quantity and quality of your sample DNA for library construction. Gel images that do not contain the appropriate size standard will not be approved for shipping. Gel images are limited to 0.9 MB in size and must be in an image format.
We require at least 30µL of DNA at 50-100 ng/µL as quantified by an intercalating dye, such as PicoGreen. We cannot guarantee results for samples at concentrations less than 10 ng/µL. Lyophilized DNA is also acceptable, but we would prefer to have DNA suspended in TE (see below). DNA concentrations do not need to be standardized across the plate; we will do this after they are received.
Samples must be supplied to us in semi-skirted or non-skirted 96-well PCR plates similar to these: Eppendorf twin.tec, semi-skirted plates, # 951020303 with caps from ABGene# AB-0783 (or equivalent). The plate must be legibly labeled with date, plate name and project number (provided after the sample approval). All plates must contain 95 samples and 1 blank, labeled “blank”. The blank must be placed in a different random well in each plate. Partial plates will not be accepted.
Approval is required before shipping samples. If samples that are shipped without approval are rejected, they will be discarded or returned, and will incur storage and/or return shipping and labor fees. In order to avoid delays and additional costs, you must wait for the formal approval email prior to mailing us samples.
Once approval is received, the shipping address will be provided. DNAs should be dissolved in TE (10 mM Tris, EDTA 0.1 mM, pH 7.5 - 8) and shipped either on dry ice (preferred) or frozen gel packs, making sure to wrap plates in bubble wrap or other cushioning to prevent plate damage. Use caps or heat seals to seal the plate. DO NOT USE STICKY TAPE SEALS. Remember to include your submission number, written both on your plates and printed on paper inside the box!