Services | DNA Sequencing | Large Templates (Lambda, BACs, PACs, YACs)

Large DNA templates (> 20kb) are routinely used in molecular biology, particularly when examining large inserts of DNA within host plasmids. These may include bacteriophage Lambda DNA (>48kb), and plasmids such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and P1-derived artificial chromosomes (PACs).

As sequencing of these large fragments of DNA can be challenging, we encourage you to review the information below prior to submission so that your reaction has the potential to yield the most robust information for your studies.

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Materials Required Per Reaction

Template: 500-1000 ng plasmid in 30 ul water per reaction

Primer: 7.5 uM in water

You should dilute your templates in water, since EDTA in TE buffer can inhibit the sequencing reaction.

When sending primer separately, please send a minimum of 10µl of 7.5 uM primer stock (to avoid issues associated with evaporation) with sufficient primer for 2µl per reaction.

Primers

Special Considerations

DNA Quality

High quality plasmid DNA is essential to achieve the best results. We would recommend a good quality mini-prep kit (e.g. Promega Wizard SV or Qiagen) or maxi-prep DNA. Large templates are difficult to purify in either sufficient amount or purity to give very good DNA sequence. Please ensure that you follow any kit instructions carefully to obtain the best yield and purity of template. It is also very important with these templates to quantify them before sending to us. However, if you would like to save time, we are happy to take care of this step for you. Just click ‘Quanti-it’ on the sample submission page.

Read Length

There are many factors that can affect the read length one obtains. Our facility will endeavour to provide customers with the longest read possible depending on service requested. Our new ABI 3730 sequencer uses the latest technology and provides really excellent results.

With a template of appropriate quality, customers can expect read lengths of up to 1000 bases of sequence from a single primer when sequencing standard templates (<20kb). For other types of template (especially for large templates), read lengths can decrease to issues of template quality and quantity. If you have problems achieving good read lengths with high quality template, please take a look at our resources section. If you still need more help, please feel free to contact us.

Pricing

Reactions

Services Requested Pricing (per Reaction)
Sequencing  £8.00
Sequencing + Quant-It* £9.00
Sequencing + Augmented Protocol^ £8.50
Sequencing + Quant-It* + Augmented Protocol^ £9.50

 

  • *Quant-It: We are happy to quantitate the concentration of your template for only £1. Please note that when sending samples for quantifying, we require a minimum volume of 10ul of material. This is because we have to use some for the quantification and if too little is sent, there will not be enough left after quantification.
  • ^Augmented Protocol: If your template has known secondary structure or a high GC content, £0.50 will be applied per reaction to facilitate the sequencing reaction using an 'Augmented Protocol'

Do you have a great deal of large template sequencing to do?

Why not contact our staff to learn about savings options?

 

Primer

  • Standard primers offered by MRC PPU DNA Sequencing & Services are free!
  • Custom primer synthesis prices will reflect the size of the primer – the cost to the customer is £0.50 per base

Learn more about all our pricing and turnaround.

Please note that prices are exclusive of VAT, which customers outside The University of Dundee will be charged at the appropriate rate (currently 20%) on all products and that all sales are subject to MRC PPU DNA Sequencing and Services' Terms and Conditions.

Turnaround Time

Given the specialised nature of these samples, please understand that turnaround times may be slightly longer than the 24 hour time-frame that is achieved for standard-sized templates.

The reason for this is that to achieve optimal results with such large templates, we have to modify our standard sequencing conditions and so these samples have to be processed separately. We routinely perform the cycle sequencing reactions on the day the samples are supplied to us, followed by evaluation of productions on our sequencers.    

Shipping

Shipping Details

  • Labels
    • Please ensure that samples are clearly labeled so that we can identify them.
  • Packing Materials
    • Eppendorf Tubes
      • Please also ensure that your samples are well protected and packaged to meet Post-Office or courier regulations
        • A "Jiffy" style padded envelope for DNA samples in plastic tubes works well
        • We discourage use of regular envelopes as in the past, we have received envelopes with tears and/or crushed tubes in them

 

Postage Costs

  • Customers are responsible for paying all postage / carriage charges
    • For DNA samples or agar plates, normal first class post should be sufficient
      • As this does not always arrive as "next day post", we recommend that customers who have deadlines to meet send their samples by a guaranteed next day delivery service
  • Insufficient postage provided on a package will cause a delay in use receiving the package
    • If MRC PPU DNA Sequencing and Services is then charged upon receipt, please note that we will need to pass this cost onto the customer

 

Postal Address:

DNA Sequencing and Services
Medical Sciences Institute
School of Life Sciences
University of Dundee
Dundee
DD1 5EH
UK

Telephone:

(01382) 388019

Fax:

(01382) 388729

Email:

info@dnaseq.co.uk

Do you need any help? Please get in touch and we’ll be happy to lend a hand.