Preparation of Samples

Overview

Sample requirements for Sanger sequencing can be found in the table below.

Process Standard PCR   PCR Clean-up Colony PCR  
Submission types Tubes or Plates   Tubes or Plates Tubes or Plates  
Sample submissions Prepped sample – note: try to avoid submitting gel extracted samples if possible Prepped sample – note: try to avoid submitting gel extracted samples if possible Liquid Culture or Extracted gDNA  
Requirements PCR Product 5µl per reaction at 10ng/µl 20µl per sample Liquid Culture or Extracted gDNA 150µl of overnight culture provided in a 96-well plate
  Primers 5µl per reaction at 3.2pmol/µl   Colony PCR Primers
100µl at 10pmol/µl per plate
        Sequencing Primers
300µl at 3.2pmol/µl per plate
Turnaround time Data delivered by 9am next day   Data delivered by 9am next day
Orders received before 5.30pm by 9am
  
       
Orders received after 5.30pm within 24hrs
  
Process Standard Plasmid   Templiphi  
Plasmid Extraction
  
Submission types Tubes or Plates   Tubes or Plates  
1.5ml Tubes or Deep well Plates
  
Sample submissions Extracted Plasmid DNA  
Extracted Plasmid DNA
   1ml of overnight culture (16-18 hours growth recommended) provided in a 96-well deep well block – preferably shaken with a breathable seal whilst incubating  
Requirements Plasmid DNA 5µl per reaction at 100ng/µl Plasmid DNA 5µl per reaction Sample 1ml of overnight culture or dried pellet
  Primers 5ul per reaction at 3.2pmol/µl Primers 5µl per reaction at 3.2pmol/µl Primers 5µl per reaction at 3.2pmol/µl
Turnaround time Data delivered by 9am next day  
24hrs from receipt of order
  
Orders received before 5.30pm by 9am
  
         
Orders received after 5.30pm within 24hrs
  
Process Premixed Sanger Run Only / Preseq Sanger Run Only Clean-up / Preseq-CL
Submission types Tubes or Plates Tubes or Plates Tubes or Plates
Requirements 5µL of DNA and your primer mixed together in equal quantities. Total 10µl per reaction. Cycled and purified reaction to be sent in dehydrated 10µl of the cycled reaction
Comments Unable to process repeat requests due to inability to check and optimse samples Unable to process repeats. Unable to process repeats.
Turnaround time Data delivered by 9am next day Data delivered by 9am next day Data delivered by 9am next day
Process Genotyping + Prep Genotyping Nanopore
Submission types Plate only Plate only Tube and Plate submissions
Requirements 5µl of diluted samples 10µl of prepared sample (inc Size Std + Hidi)

NanoporeAmplicon
15ul @30ng/μl per sample

Nanopore30
15ul @30ng/μl per sample

Nanopore150
30ul @30ng/μl per sample

Nanopore300
50ul @30ng/μl per sample
Comments We add the specified Size Std from list and Hidi Run only, no repeats. Run only, no repeats.
  Run only, no repeats.    
Turnaround time Orders received before 5.30pm same day Orders received before 5.30pm same day Data within 12 hours of sample receipt.
  Orders received after 5.30pm by 9am Orders received after 5.30pm by 9am  

DNA concentration needed

  • Plasmid: We require a concentration of 100ng/µl for plasmid samples.
  • Purified PCR samples: We require a concentration of 10ng/µl for PCR samples.

Primers

We require your primers to be sent at 3.2pmol/µl and require 5µl per reaction. You can choose to send your own primers, use our stock primers or you can order custom primers that will be delivered to the sequencing laboratory.

We recommend the following when designing your primers:

  • Choose a suitable primer length (~18 - 23 base pairs)
  • Design your primers at least 50 bases upstream of your area of interest
  • Design primers with a tm between 55°C and 60°C
  • Ensure your primers have a GC content between 40 and 60%.

Quality of sample

All samples must be of a good quality, free from contaminants such as salts, other contaminating DNA, unincorporated dinucleotides (in PCR reactions) and other reagents that would interfere with the reaction.

Prior to submission, we advise running your samples on agarose gels to ensure only one clearly defined band is visible on the gel (i.e. that your DNA is not contaminated with other DNA/it has not degraded).

It is important that you are aware of any motifs or secondary structures that might impede high quality sequencing results. If you know that this is most likely going to be a problem, we recommend you use our secondary structure resolution option when ordering.

Receiving results

You will receive an automatic email with a link to your results. This will be followed up with an email from our sequencing team the following working day. This email will include the results of their quality checks on your samples, and should samples fail, the sequencing team will advise you as to reasons why they failed and potential solutions.