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DBS Genomics

 

FRAGMENT ANALYSIS

 

Fluorescently labelled DNA fragments are detected using our Applied Biosystems 3730 DNA Analyser. Up to five different coloured fluorescent dyes can be detected in any one sample. One of the dye colours is reserved for use as a size standard, which is added to each sample to create a standard curve. The size of each dye-labelled fragment in a sample is determined by comparison with the standard curve for that specific sample.

Primers can be labelled with a number of different dyes. We are currently able to run the following dye combinations:

Filter Set Blue Green Yellow Red Orange
DS-30 D 6-FAM HEX NED ROX
DS-33 G5 6-FAM VIC NED PET LIZ

Size Standards held in stock are ROX500 (for filter set DS-30) and LIZ500 (for filter set DS-33), which cover fragments in the size range 50 to 500 base pairs.  For each filter set, only the dyes indicated can be used.  It is not possible to mix and match dyes between different filter sets.  Higher throughput can be achieved by multiplexing different dyes and fragment sizes together, however you should ensure that overlapping loci are amplified using primers with different colours of fluorescent label.  Fluorescent dyes are detected with different efficiencies, therefore PCR products should be multiplexed together at a ratio which provides similar fluorescent intensities across all loci in the multiplex.  Blue fluorescent labels tend to produce the highest signal intensity, followed by green, yellow, then red.

How to send your samples

We accept either 48 or 96 samples per plate. If using only half the plate, please ensure that samples are loaded in odd numbered wells only. Plates must be compatible with the Applied Biosystems 3730 DNA Analyser. Please contact us for advice.

Samples should be pooled into wells at an appropriate pooling ratio and dilution in order to achieve similar fluorescent intensities across all loci in the pooling. The aim is to achieve data with peaks between 500 and 20000 fu (fluorescent units). We recommend that you run a test plate initially in order to determine optimal DNA concentrations. It is a good idea to test a combination of pooling ratios to determine which ratio gives similar fluorescent intensities across all loci in the pooling, along with a dilution series of each pooling in order to determine the optimal signal strength. A suggested dilution series would be 1:10, 1:25, 1:50 and 1:100, for each pooling ratio.

Samples should be provided in a volume of 3-5ul, and the plate sealed very carefully using either adhesive film or strip caps. Plates should be packed in a padded envelope along with your completed Requisition Form, which can be downloaded from the box in the right hand sidebar. Please note that incomplete requisition forms may lead to a delay in processing your samples. Turnaround time is normally 24 hours from receipt of samples.

Samples will be labelled according to their well position on the plate. If you prefer to supply sample names then please download the appropriate Fragment Analysis Plate Record from the right hand side bar, depending on whether you are running a full or a half plate. Sample names should be entered into the Sample Name column by replacing the asterisks with your chosen sample name. Please note that the following characters cannot be used in sample names: \ / : * " < > | ? ', SPACE

The completed Fragment Analysis Plate Record should be submitted to us by email.

Receiving your data

Your data will be uploaded to an ftp site and you will receive an email informing you that the data is available and how to access it.  If there were any problems with your results you will be informed by email and offered advice as to the possible cause and how best to rectify it.  All data is archived, so can be retrieved at a later date should this become necessary.  The data provided is in .fsa format, which can be viewed and analysed with a variety of software, please refer to the software links in the right hand sidebar.

CONTACT US

DBS GENOMICS
Department of Biosciences
Durham University
South Road
Durham DH1 3LE
 
Tel: +44(0)191 3341256
 
Email: DNA.Sequencer@
 durham.ac.uk