Introduction   |  Sample preparation   |  First timer's walkthrough
Submission process   |  Troubleshooting   |  Price detail

This service begins with the enzymatic degradation of PCR primers and dNTPs from a completed PCR reaction. A single sequence is then generated from one of the primers used in the PCR amplification or a different primer that anneals within the amplicon. For best results the amplification must be free of unspecific products (you must have a single band on the gel). Please note that because the single strand specific Exonuclease I enzyme is used in the cleanup process, this service cannot be used for sequencing single stranded templates from asymmetric PCR reactions.

TEMPLATE:
For each sequence, prepare the template (PCR product) in a single well of an 8-tube strip or 96-well plate. You cannot use the conventional 1.5ml microcentrifuge tubes to submit samples for this service. Please note that if multiple sequences are to be generated from the same template you must submit that template multiple times. You will need to provide 5 ng for each 100bp of PCR product (a 600bp product requires 30ng to be submitted for sequencing) in a total volume of 12ul.
PRIMER:
Each primer to be used for sequencing must be submitted in a well of an 8-tubes trip at a concentration of 3.5uM. You will need to submit at least 10ul of primer + 2ul for each sequence beyond the first to be performed with with that primer (6 PCR products sequenced with the same primer will require a submission of at least 20ul of primer in a single well). The facility can add universal primers to any sequencing sample. In this case, you would need to submit only your custom primers to be used in the sequencing.

Our automated protocol requires the use of plasticware (tubes, strips & 96-well plates) of uniform dimensions that we provide at no charge. You can find the plasticware at any of our drop-off locations near the barcoding station. The use of plasticware with improper dimension will delay the processing of your samples.

Prepare templates in 8-tube strips or 96-well plates. Prepare primers in separate 8-tube strips even if your template strips are not filled up.

PRIMER:
Make a 20uM stock of your primers. From this stock, make a 3.5uM working solution for sequencing purposes. Submit 10ul+2ul for each sequence beyond the first to be performed with this primer into a single well of an 8-tube strip. If you need to convert from ng/ul concentrations to molar concentrations, you can use our Primer Concentration Calculator.

TEMPLATE:
Run 5ul of PCR product on an agarose gel and use a DNA size ladder with known quantities in each band. Eyeball the amount of DNA in the 5ul of loaded sample by comparing its intensity to the intensity of the nearest band in the ladder. Derive the concentration of DNA in your sample and use the appropriate amount (5ng/100bp of PCR product) up to 12ul. Note: quantitation of small PCR products by spectrometric analysis (OD260*50) is not recommended. The most precise quantitation method for PCR products is the PicoGreen fluorometric analysis; however, the sequencing reaction does not require such an accurate quantitation in order to be successful.
Bring up the appropriate amount of template to a total volume of 12 ul with molecular biology grade water.

• Use the username and password we provided you when you registered your account and log in to the system from any page of the website.
• Select the online submission form and choose the SEQUENCING submission type.
• The prompts will guide you through the process. You will need to have the following information to fill out the electronic form: name of submission, type of plasticware in which the samples will be submitted (tubes, strips, plates), names of samples and primers and how many of each you are submitting as well as which samples will receive facility provided universal primers (if any).
• Make sure to hit the ACCEPT button at the end of the process to complete your submission.
• You will receive an email with an invoice as a PDF attachment for your records.
• Proceed to the delivery of samples to one of our drop-off locations (CCHMC or GRI) and register them using the barcode scanner as indicated in the how to submit section.

-too little template
-too much template or primer
-structure in the sequence or high GC content
-multiple templates
-very long primers (ExoSAP-IT resistant) or a lot of primer in PCR
-contaminants of sequencing reaction found in high concentration in PCR

Academic: $12
Commercial: $17

Academic pricing includes CCHMC, University of Cincinnati (including the GRI), other colleges and universities and governmental orginizations such as the US EPA.