Sequencing

The MBSU sequencing facility has MegaBACE1000 (96 capillary) sequencer that uses Big Dye (Applied Biosystems) and ET-Dye Terminator (GE Heathcare) chemistries.

The reaction chemistry is based on the Sanger's dideoxy method. Each dideoxynucleotide contains a specific fluorescent dye that can be excited by a laser. The signals are then collected and analysed by specific software and coloured electropherogram is produced. The data can be remotely accessed from our secure server.

If you do not want to prepare your DNA we can do it for you, and you will receive back all the left over DNA after templiphication. We can start from a colony or a low concentration prep and amplify DNA using the Templiphi kit (GE Heathcare); the DNA Sequencing Order Formand protocols are available here to download.

Please get in touch with us if you require a quote on a specific sequencing project. Most preparations of clean template DNA produce a read of around 600 bases, including 400 to 600 bases of accurate sequence. The length of accurate sequence is primarily determined by the quality of the template DNA and the efficiency of primer annealing.

Fluorescent genotyping service

The service uses an ABI 377 and a MegaBACE1000, both with 96 sample capacity. Please download an Genotyping Order Formin order to use the service.

We can provide different levels of service:

a) Optimisation of the PCR
It is advisable to run an optimisation gel with each new labelled primer. This will enable you to get the best results from your samples.

b) Genescan samples
All Genotyping samples will be loaded by MBSU staff. The size standard will be supplied by MBSU.

c) Analysis of the samples
Using Genotyper or Genescan Analysis (for MACs) the correct sizing of your samples could be performed by us. The MegaBACE uses a software called Genetic Profiler (for PC, needs OS: Windows 2000).

d) Combinations of the above
We routinely use ET-Rox size markers from Amersham Biosciences and Rox and TAMRA from Applied Biosystems.
If required we can also give assistance (short training sessions) on using Genotyper, Genescan Analysis and Genetic Profiler as well as information on where to buy additional user licences or further copies of these programmes.