Introduction
|
Sample preparation
|
First timer's walkthrough
Submission process
|
Repeated Samples
Introduction
This service generates the sequence of a purified PCR product. For the sequencing reaction you can use one of the primers used during the amplification or a different primer that anneals inside the PCR product.
The PCR product must be purified to remove PCR primers and dNTPs. This can be done using one of several methods:
- Silica matrix capture column (like Qiagen's QIAquick)
- Excision of the specific band from a gel
- Enzymatic digestion of primers and dNTPs (like USB's ExoSapIT)
- Silica coated magnetic beads purification (like Promega's Magnesil)
Sample preparation
For each sequence, prepare the template (cleaned PCR product) and the sequencing primer mixed together in a single tube (or single well of an 8-tube strip or 96-well plate).
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) together with 7 pmol of the sequencing primer in a total volume of 12ul.
The facility can add universal primers to any sequencing sample. In this case you would simply prepare your template in a total volume of 10ul without adding any primer.
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.
Acceptable vial types for the robotic liquid handler are 1.5 mL centrifuge tubes, 0.2 mL 8-strip tubes with a single, removable cap and fully-skirted V-bottom 96-well microplates. Other vial
types (0.5mL centrifuge tubes, 8- or 12-strip 0.2 mL vials with individual caps or non-skirted pcr plates) will not be accepted. To help ensure the proper identification of the orientation of samples within an 8-strip vial, we ask that these strips not be clipped down to fewer than the 8 original wells present in the vial.
First timer's walkthrough
Prepare your sequencing sample by mixing together in a single tube (or well) primer, template and water in a total of 12ul.
TEMPLATE:
Run 5ul of purified 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 to arrive at a final quantity of 5ng/100bp of PCR product. 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.
WATER:
Bring up primer and template mixture to a total of 10 ul with molecular biology grade water.
PRIMER:
Make a 20uM stock of your primers. From this stock, make a 3.5uM working solution for sequencing purposes. Use 2ul (7pmol) in each sequencing
reaction. If you need to convert from ng/ul concentrations to molar concentrations, you can use our Primer Concentration Calculator.
Submission process
• 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.
• Proceed to the delivery of samples to our drop-off location (1st floor of the 'R' building) and register them using the barcode scanner as indicated in the how to submit section.
Repeated Samples
The CCHMC Genomics Sequencing Facility will repeat several low-quality, or 'failed' samples from nearly every Sanger Sequencing run for our own internal Quality Control purposes. Samples to be repeated are selected in a quasi-random fashion and no additional cost is incurred for these repeats by the requesting investigator. Seqeuncing data from the repeated sample is compared to the original and evaluated for quality. Nearly all repeated samples should produce similar quality metrics as the original sample from the original run. If you have a sample that has been repeated, you will receive an additional set of data files (.ab1 and .seq) for the repeated sample, designated as repeats by adding the suffix '_R' to the sample name in the files. For additional information about Sanger repeats, please refer to our Repeat Policy, found here.