Submitting plated DNAs for genotyping
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 our specifications and/or have questions or concerns, please contact our team via the “Conversations” link under the User menu.
1. DNA amounts
We require 30µL of DNA at 30-100 ng/µL (as quantified by fluorometry). We will quantify all samples arriving in the lab and inform you if the majority of samples do not meet concentration/ quantity requirements. Samples with concentrations > 100ng/ µL will need to be diluted before processing. DNA concentration does not need to be standardized across the plate but large scale adjustments to sample concentration (dilution or concentration) in our lab will incur extra processing fees. We will process samples below 30ng/µL, but we discourage submission of these low concentration samples. Samples at concentrations less than 10 ng/µL often do not produce adequate numbers of DNA sequences.
2. DNA quality control
Trial digestions of at least 10% of your samples must be performed as follows: digest 300-500 ng DNA with 3-5U (1U per 100ng DNA) of a restriction enzyme that is NOT methylation sensitive (i.e. HindIII or EcoRI) and run on 0.8-1% agarose gels along with the uncut sample DNA (100ng) and 500ng of λ HindIII size standards. 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 be accepted but we will not be able to estimate sample concentrations from the gel image.
Image requirements: Gel images are limited to 0.9 MB in size and must be in an image format (jpg, gif, tiff…. not pdf). There is also a limit of five images per template uploaded. Please label your samples and size standards clearly.
3. Submitting sample information
Using the “Create Plate” button at the bottom of your project page, download a sample submission template for each plate to be submitted. Fill in the required fields (sample name, DNA concentration, DNA volume and DNA mass) and upload the completed template and associated DNA QC gel image(s). Please note that the LIMS assigns the position of the empty well on each plate. Therefore, do not load DNA samples onto plates before downloading the sample submission template.
Version 2 Updated 2016-02-12