DNA Sequencing Services

What services do you offer?

We offer Sanger DNA sequencing, PCR, Plasmid Purification, Picking & Growing Colonies, Primer Walking, Capillary Electrophoresis on already sequenced and cleaned up samples (Ready to Inject Plates) or on samples already sequenced but still need clean up.

What is the turnaround time for results?

The turnaround time depends on the order type and number of reactions.

For individual sample orders sent as purified DNA or crude PCR product, the turnaround time is normally 24 hours if samples are received by 4:00pm. Re-sequencing reactions for any reason adds an extra business day (not including weekends).

For plate orders the turnaround time can take anywhere from 2-3 business days, however most plate orders are completed in 2 business days. For plate orders containing a large number of plates the turnaround time will be a little longer, but we will provide you the results as we get them done which should start within 72 hours.

It will take one extra day for samples sent as colonies on agar plates for plasmid purification or for pelleted cultures.

What machines do you use?

We use the ABI 3730xl DNA Sequencer with 50cm arrays.

What is the average read length per sample?

All of our reads typically read out to over 1000 bases; however, we usually get approximately 800 high-quality bases per reaction. The read length will vary depending on sample quality and type.

Sample Requirements

What concentration should my primer be?

All primers should be sent at 10µM.

What volume of primer should I send?

For individual samples, a minimum of 10µl should be sent. If you are using the same primer on multiple samples, we require just 3µl multiplied by the number of reactions you are doing with that primer, but still a minimum of 10µl.

For full plates we need 100µl of primer at 10µM per plate. If you are sequencing multiple plates that will use the same primer, send 100µl plus 30µl for each additional plate.

If you are submitting a primer plate, include at least 10µl of primer at 10µM per well of the primer plate.

Can I premix the sample and primer?

Yes. Be sure you have a total of 20 picomoles of primer per 15µl of premix. We recommend using the following formula to create pre-mix samples:

DNA: N=0.1 x (vector size in base pairs + insert size in base pairs)

*N is the nanograms of DNA that should be added to each 15µl of premix.

What should the sample concentration be?

The sample type will determine the concentration needed.

i. PCR Product: 25-50 ng/µl

ii. Plasmid DNA: minimum of 50 ng/µl but we prefer it at 100 ng/µl. If you are sending it at 100ng/µl we need 10µl for one reaction or 15µl for two reactions. If you are sending it at less than 100ng/µl please send 15µl for one reaction or 25µl for two reactions.

*Note: If your samples are below the desired concentrations as listed above, we can still sequence them, the results are just less likely to be of high-quality. Please indicate the concentration on your order form if known so that we can adjust the sample volume to add per reaction accordingly.

I am sending cultured pellets and am unsure if they are sufficient. Do you have any suggestions?

If you can check the O.D. of the solution before pelleting, it should be around 3.0-4.0. If you did not check the O.D. prior to pelleting , feel free to send the pellets and, if upon arrival they appear too small, we will contact you and inquire about re-growing your samples here at our facility if needed.

*If we have to re-grow them this will add an additional day for turnaround time.

Do I have to send my own primer?

No. We have a list of free primers. We can also synthesize and/or design your primers for you. Primers are stored for a period of 2 years.

What primers do you offer for free?

Please refer to the primers page on the website for a complete list.

Can you do primer synthesis?

Yes. It is $16 per primer for standard primers. If you want us to design and synthesize the primer the cost is $25 per primer.

Can you quantify my samples?

Yes, if you would like any of your samples quantified, please indicate which samples you would like us to quantify in the notes section of your order form. We CANNOT quantify crude PCR products, or premixed samples.

Sample Submission

Should I print out my order and send it with the samples?

Yes, please!

Can I modify my order after it has been submitted?

No, you cannot. If you have not yet sent your samples you can record changes to the order on the copy of the order form you printed out and are sending along with the samples. If you already sent the samples you can email us at sequence@functionalbio.com to explain the change or call us at (608)441-8125 / (877)293-7947.

When creating my sample submission form, I would like to attach a file indicating specific instructions. Would you prefer this in a specific format?

For full plates use the full plate template you can save a spreadsheet for each plate you are sending and then upload it in the box next to the plate name on the order form. Any additional notes or information can be submitted in the notes section of your order form.

*Please be sure not to use any characters other than letters, numbers and dashes/underscores.

What if I already submitted my order and forgot to include a spreadsheet?

If you already submitted the order feel free to email us a copy of the spreadsheet, sequence@functionalbio.com, and indicate which order it should go with.

Can I cancel my order?

Yes. If you do not send the samples, we will not process the order.

Which carrier should I use for shipment?

Whichever is most convenient for you; we recommend UPS or FedEx for faster shipping. It is best to send the samples priority overnight so we get them by 10:30 a.m. If you send them standard overnight, we may get them in the morning but sometimes we do not get standard overnight shipments until after 3 p.m. which can delay the results. If you choose to send samples USPS, please understand that there may be an extra day for turnaround as we do not receive those packages until late in the afternoon.

Can I request a custom annealing temperature for my samples?

Yes, but requesting custom annealing temperatures will cause a delay in sequencing results. Note that a PCR annealing temperature does NOT need to be the temperature the sequencing reaction is run at.

Sequencing Results

How do I receive the results?

We will send you an email when your results are posted to the website. You can then login and download your results.

Will you keep my samples and/or primers?

We will hold on to any samples and primers sent with an order for approximately two months unless otherwise specified.

If we synthesize a primer for you, we will put it in our long-term storage which is kept for two years. If requested, we can hold onto it for longer while replenishing the stock as needed.

Can I get my samples back?

Yes. We will ship them to you if requested; however, we will add the shipping charge to your invoice for this service.

Can I submit a new order and request to use samples I previously sent to you?

Yes. Please indicate in the notes section of your order form if the samples or primers come from a previous order. You can call or email to be sure we still have the samples you are requesting at a sufficient volume.

How long are results available on my account?

Results are available for six months from the date they are posted.

What are the 5 txt files that come with my results?

Below is an explanation of each type of file we provide.

.ab1 Files: The .ab1 file is the chromatogram (trace) file from your sample. By viewing this file type you’ll be able to see the actual peaks associated with each base call. Programs to use: There are a number of different programs that can be used to view these files. We have links to a couple of these, here.

.raw.seq.txt: This is an untrimmed fasta file. This file is not edited and may contain some low quality base calls. It’s important to verify these calls by viewing the .raw.qual.txt quality score file or the peaks in the chromatogram.

.raw.qual.txt: This file lists the phred quality score of each base in the .raw.seq.txt file.
Probability of Error = 10^(-Quality Score/10)
So a quality score of 20+ means there’s less than a 1% chance of error.

seq.txt: This is the trimmed version of the fasta file. In this file, high quality bases with phred scores above 20 are capitalized, low quality bases are in lower case. Low quality bases are trimmed off the start and ends of the sequences. Wells that have no high quality bases are omitted from this file.

seq.qual.txt: This file lists the phred quality score of each base in the trimmed seq.txt file.

Seq.info.txt: This file lists information on read lengths and quality scores for each well.

Billing

What are my payment options?

You can pay by check, credit card, wire transfer, or direct deposit. We can also set you up with a pre-paid account or automatic payments.

When can I expect to be invoiced?

Invoices are sent out shortly after the order is completed; typically within one to two weeks.

Do you require a PO on all orders?

No, we do not require a PO. This is up to your institution. If you are required to have a PO by your institution, please indicate the PO number on your sample submission form along with the email address the invoice should be emailed to.

How are invoices sent?

All invoices are sent via email. Please ensure to add the correct billing email address when you setup your account.