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DBS Genomics

 

DNA SEQUENCING 

 

Our DNA Sequencing Service is quick and easy to use, with no need for lengthy registration or passwords to remember. Simply download a requisition form, complete and send with your samples.

Samples and primers may be sent by post or overnight carrier. Lids should be secured with a little sealing film, and packed in a padded envelope along with your appropriate 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. Please provide as much information as possible about your samples as this can often influence the way we process them, for instance in the case of Gateway vectors which contain palindromic sequences and are prone to secondary structure problems or samples with a high GC content. Turnaround time is normally 24 hours from receipt of samples.

Upon receipt, your samples will be run on agarose gel to assess quality and concentration. Sequencing reactions can then be carried out using conditions optimal to each individual sample, ensuring the best data possible is obtained for each reaction. All data produced is evaluated with some manual editing where necessary. Your sequence data will be returned to you by email, or large batches will be uploaded to an ftp site. 

We place a high emphasis on customer service and will provide tailored troubleshooting for any problem templates, and advice on sequencing strategies. For pricing information please follow the link in the right hand sidebar.

Internal users should refer to the Internal Users' Guide in the Related Links box in the right hand sidebar, for guidelines on using the service.

How to send your samples

We accept single tubes and half or full 96-well plates, either as DNA samples or completed sequencing reactions which are ready to load. 96 well plates must be Applied Biosystems 3730 compatible (please contact us for advice if needed). If only half a plate is used, samples should be loaded into odd numbered wells only.

 

Single Tubes

We ask for a volume of 6µl of DNA per reaction to be sent so that we have sufficient to check the sample on an agarose gel and still have enough left to carry out the sequencing reaction even if the DNA is weak.  If you have more DNA to spare please send it so that we have some available should we need to repeat any reactions.  For advice on DNA preparation, please follow the link in the right hand sidebar.

Custom primers, if required, should be sent at a concentration of 3.2 pmol/µl, a volume of 3µl per reaction is sufficient.  We hold a selection of universal primers in stock, for details of these and advice on custom primer design, please follow the primer information link in the right hand sidebar. 

Samples should be packed in a padded envelope along with your completed requisition form.

 

Plates

For plasmid samples we require 125ng of DNA in a volume of 5µl of water to be added to each well, along with 1µl of primer at a concentration of 3.2 pmol/µl.  For PCR products we require 5ng of DNA per 100bp of product, in a total volume of 5ul.  (For example, for a 500bp PCR product with a concentration of 50ng/µl, we would add 0.5µl of DNA to 4.5µl of water + 1µl of primer ). We are able to run full or half 96 well plates.  If using only half a plate please load samples into odd numbered wells only.  If you would like to supply sample and primer names you should download and complete the appropriate Plate Record for either full or half 96 well plates.  Sample/primer names should be entered in place of the asterisk at the appropriate well position, and the Plate Record submitted by email.  If no sample names are provided then samples will be labelled according to their well position.  Please note that the following characters cannot be used in sample names:   \  / : * " < > | ? ' SPACE 

Plates should be sealed carefully and packed in a padded envelope, along with your completed requisition form.

Ready-to-load reactions

If you prefer to prepare your own sequencing reactions we are able to run either individual tubes, half plates or full plates.  If only half a plate is used then samples should be added to odd numbered wells only.  Sequencing reactions should be carried out using Applied Biosystems BigDye Terminator Ready Reaction Mix, followed by an appropriate cleanup method.  If you would like to supply sample and primer names you should download and complete the appropriate Plate Record for either full or half 96 well plates.  Sample/primer names should be entered in place of the asterisk at the appropriate well position, and the Plate Record submitted by email.  If no sample names are provided then samples will be labelled according to their well position.  Please note that the following characters cannot be used in sample names:   \  / : * " < > | ? ' SPACE  

Samples should be packed in a padded envelope and submitted along with your completed requisition form.

 

Primer Walking

Template DNA can be sequenced on one or both strands in its entirety. We generally initiate sequencing from both ends of the template and stop either at the overlap or when both strands are complete depending on the request. Occasionally, sequencing from one direction only is possible if there are problem regions on the template.  In extreme cases it may be impossible to produce a complete sequence due to regions of secondary structure, repeats or other difficult regions of sequence.

 

The primer walking service includes:

  • Single or double stranded sequence generated
  • Custom primer design and synthesis
  • Manual editing of all data
  • Final assembly of complete sequence

We require approximately 0.25μg of DNA per Kb of single stranded sequence.  We hold a selection of universal primers in stock, for details of these please refer to the Primer Information in the right hand sidebar.  Custom primers, if required, should be sent at a concentration of 3.2 pmol/µl, a volume of 3µl is sufficient.  Samples should be packed in a padded envelope along with your completed requisition form.

Receiving your data

Your data will be sent to the email address provided on your requisition form, as a simple text file with chromatograms (.ab1 files) attached.  Large batches of data will be uploaded to an ftp site and you will receive an email informing you when the data is available and how to access it.  If there were any problems with your sequencing you will be informed by email and offered advice as to the possible cause and how best to rectify it.  Some commonly occurring problems are outlined in a troubleshooting guide, follow the link in the right hand sidebar.  All sequence data is archived, so can be retrieved at a later date should this become necessary, and all DNA samples and primers are retained for up to one year.

There are various freeware programs available which can be used to view and edit your sequence data.  For more details follow 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