Volume
We need 5µl per reaction of both your primer and DNA at the concentrations specified below:
| For standard Sanger Sequencing | |
|---|---|
| Plasmid DNA | 100ng/µl |
| PCR product | 10ng/µl |
| Sequencing Primer | 3.2pmol/µl |
| For Colony PCR and Bugs2Bases (plate only services) | |
| Bacterial Culture (Plasmid Extraction) | 150µl of overnight culture provided in a 96-well plate – preferably shaken |
| Colony PCR Liquid Culture | 150ul of overnight culture provided in a 96-well plate |
| Colony PCR Primers | 100ul at 10pmol/µl per plate |
| Sequencing Primers | 300ul at 3.2pmol/µl per plate |
DNA concentration needed
- Plasmid: We require a concentration of 100ng/ul for plasmid samples.
- Purified PCR samples: We require a concentration of 10ng/ul for PCR samples.
Primers
We require your primers to be sent at 3.2pmol/ul 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.