The information below provides answers to the questions we get asked often. If you are looking for trouble shooting information (e.g. because you are unhappy with your results), please go to our trouble shooting area. Should you not find the answer to your question/problem here, please contact us and we will do our best to help.

What is automated DNA sequencing anyway?

Automated DNA sequencing is a method of sequencing DNA that uses fluorescent dyes to allow detection of the DNA fragments by a laser. The information is then analysed by a computer to generate the final sequence data. This has a number of advantages over conventional DNA sequencing:

1. Handling and disposal of radio-isotopes is eliminated.
2. Sequence length and accuracy are improved.
3. Sample throughput is much greater
4. Data acquisition is much faster

A number of platforms for automated DNA sequencing exist, but the most widely used are those supplied by Applied Biosystems. The most common type of chemistry that is used on automated DNA sequencers is dye-terminator chemistry. This makes use of linking a different fluorescent dye to each of the four different di-deoxy-terminator nucleotides. When the chain is terminated by addition of a di-deoxy-terminator nucleotide, the fluorescent dye is incorporated. This type of chemistry allows all four fluorescent markers to be present in a single reaction, thereby greatly enhancing throughput.

Dye-terminator chemistry is most frequently coupled to the use of Thermus aquaticus (Taq) DNA polymerase in a reaction known as "cycle sequencing". This makes use of the thermostable nature of the Taq polymerase to allow multiple rounds of sequencing from a single template. By doing this, the amount of template DNA required is substantially reduced.

What types of products do you provide?

We have a range of products aimed at meeting the needs of most customers. For a full list of the products we provide, please follow the products link at the top of this page. If you have a sequencing need that is not covered by our standard types of product, please feel free to contact us.

What types of DNA can you sequence?

We can sequence most common types of DNA, including plasmids, single-stranded phage, PCR products, Lambda and BAC DNA. For a list of the types of DNA we can sequence and amounts required, please follow the services link at the top of this page. For plasmids, mini- or maxi-preps can be used, although mini-preps must be of good quality if reliable sequence data is to be obtained. We use Promega Wizard SV mini-prep DNA with excellent results. Single-stranded bacteriophage DNA works well if clean, as do PCR products provided that the primers and dNTPs used for the PCR are removed! Lambda and BAC (bacterial artificial chromosome) DNA can be sequenced and, again, the quality of DNA is very important as well as the quantity. All types of DNA to be sequenced must be supplied in Milli-Q water, since Tris and EDTA in TE buffer can interfere with the sequencing reaction.

How much DNA do I need to supply?

The quantity of DNA required depends on the type of template DNA you wish to sequence. Information on the amounts of different templates required can be found on the appropriate contract research option pages. Your template should always be diluted in water so that 15µl contains the correct ng amount for one reaction. For all services except the 96 well plate service, we then ask that you send 30µl of each template per reaction you want performed, so that we have sufficient for repeats should they be necessary.

We do strongly recommend quantitating your template DNA on an agarose gel against a DNA of known concentration (for example, Lambda DNA purchased from a supplier of molecular biology reagents - we use Lambda DNA from Promega). Failure to do this will almost certainly result in an over-estimate of the quantity of DNA you have (especially for mini-prep DNA) and lead to poor quality sequence due to the use of too little template. If you have access to a Nano-drop spectrophotometer (or equivalent), you can use that for quantitating mini-prep DNA. The use of a standard spectrophotemer it not recommended, since the dilution used results in an inaccurate measurement.

How much primer do I need?

The amount of primer is template-dependent, although for most templates it is 3.2 picomoles of primer per reaction (remember that only ONE primer is used per reaction!). Details of the amount of primer required for different templates can be found on the relevant contract research option pages. Quantitation of primers is best done by spectrophotometric methods. We use the conversion factor of an OD 1.0 at 260 nm is 20 micrograms per ml of oligonucleotide in the cuvette. Oligonucleotides should always be diluted in Milli-Q water.

What types of primer work?

The type of primer that can be used for automated DNA sequencing is very dependent on the chemistry used. Since we use cycle sequencing for all our reactions, the primers must be designed with this in mind. Following a few basic rules will ensure that your primers have the correct properties for cycle sequencing:

1. Always design your primers using known/good quality sequence
2. Aim for a Tm of about 60 degrees celcius [Tm=(sum A+T) x 2 + (sum G+C) x 4]
3. Try to have about 50-55% GC content
4. Have at least one G or C at the 3' end of the primer (but not GGG, etc.)
5. Avoid homopolymeric regions (e.g. CCCCC)
6. Avoid repeats of sequences (e.g. GATCGATC)
7. Avoid any self-complementary regions (e.g. GATCNNNGATC)

Anyone familiar with PCR primer design will recognise the above rules. Cycle sequencing is similar to PCR (except only ONE primer is used) and so often primers that work for PCR will work for cycle sequencing. However, primers that have excessively high Tms (over 65 Deg. C) may generate high backgrounds, when used for cycle sequencing. Primers with low Tms (below 55 Deg. C) often give weak sequence. This can also be seen with certain "standard" sequencing primers such as T3 and SP6. For this reason these primers have had to be altered for automated sequencing.

Do I provide the primers?

We can supply certain standard primers (at no charge). However, if you intend to use a non-standard primer that is not on our list of primers, then you will have to provide your own primer. We recommend making stocks of primers at 3.2 micromolar. 1 microlitre of this primer is then used in a reaction, leaving the maximum volume possible for template DNA (important for templates at very low concentrations).

Please note that when we undertake a primer walking project, we agree to make all the necessary primers for the project, all you have to do is to supply the DNA. For more details, please see our description of this service on the appropriate service page.

How fast are samples processed?

We aim to process standard samples within 1-2 working days. For DNA samples that arrive first thing in the morning, we will usually set up the cycle sequencing reactions in the morning and then queue them on the sequencer in the early afternoon. Depending on the queue, this can mean that samples are sequenced and the data uploaded to the web site the same day! Failing this, they will usually be ready to download the following morning. DNA samples that require us to prepare the DNA, then perform cycle-sequencing are usually ready in 3-4 working days. All the above times are dependent on sample load and the more work we have to do the longer it can take if we are very busy!

How do I get my results?

All of our customers should download their results from our web server. This is the only method we now offer, since it is quick, efficient and cheap (helping us to keep our prices down). We have found that customers also prefer this method.

What read length should I expect?

There are many factors that can affect the read length you obtain. However, we always try to give you the longest read possible depending on service you have asked for. Our new ABI 3730 sequencer uses the latest technology and provides really excellent results. With a good template, you should get back up to 1000 bases of sequence from a single primer when sequencing standard templates. Our best read length to date on the ABI 3730 is 1200 bases! For other types of template (especially for large templates), read length can be less due to issues of template quality and quantity. If you have problems achieving good read lengths with high quality sequence, please take a look at our trouble shooting section. If you still need more help, please feel free to contact us.

How much does it cost?

There's always a catch isn't there! Sadly we do have to charge for the products we offer (well, we do have to eat!). We attempt to keep our prices low by offering good value for money options that should "work" most of the time. We cannot do this AND guarantee results. Hence, we will charge you even if your sequencing is not a success (unless, of course the fault is ours, in which case we will perform your sequencing again for free).

Our standard policy on failures and repeats is: Should your sequencing fail and you ask for a repeat, we will perform this using the remainder of the sample you supplied. Should the reaction work well the second time, you will not be charged for either sequence run. However, should the reaction fail again, you will be charged for both. This might seem harsh, but a consistent failure almost certainly means that the reason for failure is outside of our control and so we should not be penalised for that.

However, we would like your DNA sequencing to work first time, since it means you are happy and it means less work for us doing repeats and trouble-shooting your problems. This is one of the reasons for the support pages on this site! We also have a trouble-shooting page and are happy to answer any of your questions regarding automated DNA sequencing, especially BEFORE you give us something that doesn't work! Please contact us if you have questions.

How should I acknowledge DNA Sequencing & Services?

We all like to receive credit for the work we do don't we? We would be grateful if people publishing work that utilised our facility acknowledge the work we did. A simple acknowledgement is sufficient and can either be placed in the materials and methods section or at the end of your manuscript depending on journal format. Suggested formats are given below.

Acknowledgement in materials and methods:

DNA sequencing was performed by DNA Sequencing & Services (MRCPPU, College of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.

Acknowledgement at end of manuscript:

We thank DNA Sequencing & Services (MRCPPU, College of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) for DNA sequencing.