Sample Submission
Sample submission considerations.
The facility provides a number of standard sequencing primers for common plasmid vectors.
Custom primers should be provided at a concentration of 5 uM (=5 pmol/uL).
Request Forms:
Individual Sample Submissions
Sanger Sequencing Request - Use this form to submit DNA templates (ie. PCR amplicons, Plasmids, etc.) for sequencing
DIY Sequencing Request - Use this for to submit completed fluorescent Sanger products (ie. BigDye reactions) for sequencing
For full and half-plate submissions please contact the facility
Equipment Used: Applied Biosystems 3730 DNA analyzer
|
SERVICE |
INTERNAL |
ACADEMIA |
COMMERCIAL |
|---|---|---|---|
|
Single reaction |
$9 |
$9 |
$16 |
|
96 Reactions (Full Plate) |
$500 |
$600 |
$750 |
|
DIY Cycle Sequencing* - single reaction |
$5 |
$6 |
$8 |
|
DIY Cycle Sequencing* - up to 48 reactions |
$75 |
$100 |
$150 |
|
DIY Cycle Sequencing* - 96-well plate |
$150 |
$200 |
$300 |
|
Fragment Analysis - up to 48 reactions |
$50 |
$75 |
$125 |
|
Fragment Analysis - 96-well plate |
$100 |
$150 |
$250 |
Note:
*DIY Cycle Sequencing is performed by clients with using a Sanger Sequencing Mix (ie. BigDye Terminator V3.1 Mix) which can be purchased separately from the facility using the AAC Consumable Order Form.
FAQs
What are your recommendations for DNA concentration and quantity?
| DNA Template Size | Concentration (ng/µl) | Quantity (ng/rxn) |
|---|---|---|
| 100–500 bp | 5–10 | 20 |
| 500–1000 bp | 10–30 | 50 |
| 1 kb–5 kb | 30–100 | 150 |
| 5 kb–10 kb | 100–200 | 300 |
Please provide 3-10 µl of sample. We will accept samples outside these guidelines, but sequencing success rate will be lessened.
Do you run QC on my samples?
We do not verify the quality nor concentration of submitted samples for Sanger sequencing. Every sequencing run (excluding DIY submissions) includes an in-house control—if quality thresholds are not met, sequencing will be redone at no cost to you. For other sequencing concerns please see our troubleshooting notes below.
How long do you store unused sample?
We store unused sample for one month. Additional storage is available on request.
How long is my data stored?
Data will be stored for at least 1 month. Backups may be available on request, but we do not guarantee data availability after 1 month.
What is your sequencing turnaround time?
The typical turnaround for Sanger sequencing is 48 hours.
What can cause failed sequencing?
Failed sequencing can be caused by incorrect template concentration, poor quality DNA, badly designed primers, contamination, or machine failure (e.g. blocked capillary). To troubleshoot poor sequencing, we recommend looking at your trace files. For instance consider the following examples:
This sample features a large dye blob, a result of contaminating DNA or residual dNTPs in the sample causing a peak of fluorescent signal. In this example, the signal strength of the sample is weak, and therefore it is very difficult to determine the sequence in this region. This may indicate that not enough template was present in the BigDye reaction.
In this example, a small dye blob is still present in the same region of the spectrum, but since signal strength is much higher bases can still be accurately called in the region. If you are having problems with signal strength make sure you have accurately determined the concentration of your sample prior to submission, and that your primers are targeting the region of interest as expected.
In this example, many peaks can be seen interleaved on top of one another. This typically results from a mixed sample, if you had prepared amplicons from a taxon of interest it is possible that your sample was contaminated. It may also represent off target effects of your primers.
For further assistance with Sanger sequencing troubleshooting, please reach out to us for a consultation.
What is your policy for failed sequencing samples?
Every sequencing run (excluding DIY submissions) includes an in-house control—if quality thresholds are not met, sequencing will be redone at no cost to you.
Can you design primers?
We can provide guidance, but do not design primers. Please reach out to us for a consultation.
Which primers do you have available?
A comprehensive list of primers offered by the facility can be found here.
Can you run Sanger sequencing with my own primers?
Yes! We are able to use any primer for the reaction. Please provide an aliquot along with your samples containing a 5 nM dilution of your primer with 2 uL per reaction using this primer. You can request multiple different primers with the same sequencing request.
How will I receive my data?
We provide both raw .ab1 and .seq files from the genetic analyzer via email. For other methods of transfer, please contact us.
Do you provide any bioinformatic analyses for Sanger sequencing?
Please contact us for a consultation for any further sample processing.