Sanger Sequencing, the workhorse of the human genome project, is still the gold standard technology for sequence analysis of plasmids and PCR products.
The Genome Centre uses a third party to sequence up to 850 base pairs with a quality (Phred) score over 20. We are able to help with primer design and primer walking and contig construction, with data analysis and quality control using the Phred/Phrap/Consed suite of software developed by the University of Washington. We can also help with troubleshooting difficult-to-sequence templates.
Samples can be dropped off at the centre daily by courier or hand delivered. Users at the Blizard Institute may make use of the sample collection box that leaves from the post room there at 11am each morning, Tuesday to Friday. Data will be returned within two working days; a premium service can be arranged with quicker turnaround if required. Data is returned attached to the original iLab request.
Sequencing requests – should be submitted on our iLab service request page for which you will need an iLab login. You will need to discuss this with your lab manager.
PCR products – 5µl PCR product per sequencing reaction (80–100 ng/µl) – sample clean up using Shrimp Alkaline Phosphatase and Exonuclease I is provided. It is strongly recommended that you run your products on a gel prior to sample submission.
Plasmids - Cleaned plasmid DNA at 100ng/µl 10µl per sequence. Care should be taken quantifying your plasmid. The effects of template concentration on sequence quality can be seen in our technote: Sanger Sequencing sample primer concentration technote [PDF 524KB].
Primer requirements – Custom primers should be provided separately by the user at 5µM or 5pmol/µl. A minimum of 5µl per sequence. Some common plasmid primers are provided free of charge by the Centre.
Pre-sequenced samples – We can now only accept half (up to 48 samples) or full (up to 96 samples) plates of pre-sequenced samples. These should be prepared using ABI's BigDye Terminator cycle sequencing kit v3.1 at 10µl and should not be cleaned; this is provided as part of the service as standard. Samples should be provided in semi-skirted plates sealed with Flat Cap Strips: Thermo Scientific, item number: AB0786
Formats – For sequencing jobs of less than eight samples, individual tubes are adequate. For larger sequencing jobs we prefer samples in either PCR strips or a standard 96 well microtitre format.
Data queries – Data queries should be raised as soon as possible – remember we are here to help you get good data quality. The contract we have with our third party provider assumes that the template and primer are provided at the correct concentration. Repeats and troubleshooting for failed reactions comes at the standard rate.
Sample handling – Surplus samples and primers will be retained for four weeks before being disposed of. A request may be made to retain primers for future sequencing jobs.
To discuss projects or for costings please contact Dr Charles Mein on 020 7882 2055 or by email at c.a.mein@qmul.ac.uk.
Full details of technologies and services can be found on our services menu.