Services | DNA Sequencing | Primer Walking

Template

As with other sequencing reaction options such as Plasmid , PCR and 96-well Plates, we ask customers to provide us with their template DNA in sufficient quantities. Please provide staff with enough high quality plasmid DNA for the project. 10-20 µg of plasmid is enough for most projects (<20 kb). An accurate DNA concentration is required. Determination by agarose gel electrophoresis can suffice (including a picture of the gel), but 260/280 nm analysis by a spectrophotometer is ideal.

Please ensure template is diluted plasmid in water or a buffer that does not contain EDTA (such as Tris-EDTA buffer) which can inhibit the sequencing reaction. 

Sequence Information

Please provide as much information as possible about the plasmid/insert including:

1. Plasmid name
2. Size
3. Restriction sites used to clone the insert
4. Priming sites, etc.

Please also supply any sequence information you currently have, since it will help us to confirm that we are sequencing the right thing at the start.  

Upon customers providing MRC PPU DNA Sequencing and Services with the information requested above, we will be happy to design the requisite number of primers that will enable us to ‘walk’ through your insert and provide the required sequence information critical for your studies.

Standard Procedure

In most cases, we initiate sequencing from both ends of the insert and when the sequences overlap in the middle, we stop (or carry on and fully sequence both strands if this is what you have asked for). In some cases, we may only be able to sequence from one end (e.g. due to polyA tail on cDNA, or because of some other problem). If this happens, we will contact you right away.

The costs of all sequencing primers/reactions and assembly of the final sequence are included. You will receive back the completed sequence plus all primers synthesised for the project. Data acquisition is about 1kb a week (if sequencing from both ends) and we can also provide you with the sequence as it is generated.

DNA Length

This service is optimal for ‘walks’ of between 2-10 kb (not including plasmid backbone). If the region you wish to be sequenced and analysed lies outside this size, please contact us

MRC PPU DNA Sequencing and Services will sequence the entire insert – on one or both strands, as requested.

Analysis of ‘Walks’

Please note that we cannot guarantee to be able to complete primer walking projects, since secondary structures and difficult regions of sequence can make it challenging to read through certain sequences. However, we do try our best and if we cannot complete the project, we will just charge for the sequence that we can obtain.

MRC PPU DNA Sequencing and Services charges the following for its Primer Walking service:

• £0.15 per base (one strand/direction)
• £0.30 per base pair (both strands/directions)

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.

Details of Services

This service requires a great deal of attention to detail required for this analysis, and our facility includes the following aspects of this service within the costs:

• Strategy
  • Design of overall strategy, in agreement with customer, prior to commencement of work 
• Template Analysis
  • Quantification/qualification of template material provided
  • Dilution to appropriate concentration
• Primer Evaluation
  • Design
  • Synthesis
  • Quality control evaluation
  • Dilution  
• DNA sequencing 
  • Carry-out all reactions
    • Including optimisation of reactions as necessary
      • In cases of problematic areas of sequence, reaction conditions will be modified as needed
• Final Customer Deliverables
  • Generation of a complete contig assembly of the sequence
    • Annotated contig, primer sequences and locations
  • Primers and unused template

Given the fact that the nature of each sequence can vary in size, turnaround times will reflect the time it takes to evaluate the full sequence requested by customers. Data acquisition is approximately 1kb per week (if sequencing from both ends). Should customers request it, we can also provide you with the sequence information as it is generated by our staff.

Shipping Details

• Labels
  • Please ensure that samples are clearly labelled 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 DNA Sequencing and Services is then charged upon receipt, please note that we will need to pass this cost onto the customer