Instructions for Submitting Samples for Sequencing
Fees:
$5.00* per sample template for Internal Users (URI, RI-EPSCoR, & INBRE).
$8.00 per sample template for External/Non-Profit Users.
$15.00 per sample template for External/For-Profit Users.
* Beginning July 1, 2008, users at all Rhode Island EPSCoR institutions receive a 40% subsidy of the above fee.
Internal Users:
Use the GSC as the vendor (PeopleSoft Vendor ID = URI_GSC).
For a one-time submission, please provide a College Requisition
as payment for services when you submit your samples.
For multiple submissions, please provide an Internal Purchase
Order as payment for services when you first submit your samples. This
Internal PO should state "Blanket Requirements: start date - end date"
in the description and should cover the number of samples you anticipate will be
sequenced during the time period.
External Users:
Please provide a PURCHASE ORDER with the Genomics
and Sequencing Center as vendor as payment for services when you submit
your samples. Please also include your billing address.
Sample Preparation:
Follow the Sample Preparation Guidelines in order to optimize your template and primer
for the Applied Biosystems BigDye® Terminator v3.1 chemistry. Template purification and
quantification are the most critical factors in obtaining good sequence data. If you are
unsure of the quality of your DNA, please check on an agarose gel before submitting the sample.
We are not responsible for poor sequences due to impure templates.
Sample Submission:
Fill out a Submission Form to identify all samples. Submit your template and primer combined
in a 0.5 or 1.5 ml tube. DO NOT submit samples in individual 0.2 ml (200 µl) tubes.
A Submission Form generator is also available that will calculate the amounts of template needed for both PCR products and plasmid templates. The instructions are very easy to follow and will result in a submission form that can be printed from your web browser.
Target amounts for dsDNA templates:
- PCR products: 5 ng DNA per 100 bases per reaction
- Plasmids: 300-500 ng DNA per reaction
Primer amount:
- 5 pmol per reaction (Note: 5 pmol = 2.0 µl of a 2.5 µM stock)
Single sample volume:
- 12 µl per reaction; add template plus primer in the amounts above
to MB grade water.
To facilitate pipetting, submit your sample in duplicate with a total volume of 24 µl.
When submitting 16 or more samples, please submit them in 8-tube strip-tube(s) (capped) or a
96-well plate (capped or sealed).
If submitting cosmid, BAC, or genomic DNA templates, please call for more specific information regarding amounts.
Labeling Samples:
Please identify your samples using the following code (6 character limit): Your initials
followed by the number 1 to 9999 (i.e.: PJ1, PJ2, etc.). You should increment the number
with each submission. This code facilitates instrument plate setup and file management.
Sample Analysis:
Sequencing on the ABI 3130xl genetic analyzer is routinely conducted
using POP7 polymer, a 50 cm. 16-capillary array and the KB™ Basecaller software.
These conditions normally produce high quality sequence that extends to 800-1,000 bases.
PCR products less than 900 bp in length will be analyzed in the 3130xl
using an analytical protocol that looks for the end of the raw data. Please identify
your PCR product and its size on the Submission Form so this protocol may be specified
during instrument setup.
ALL sequence data will be examined for quality and content. Please note
that sequence data are NOT trimmed or edited!
Results:
Sequence data will be exported in the standard Applied Biosystems format,
ab1, and distributed by email upon completion of the run. Formats seq
(ABI and FASTA text only sequence), scf, and phd.1 (Phred) are also available
at the request of the user. Please provide an email address with sufficient
capacity to receive your data. Alternatively, your data can be copied to a
CD-R. You may provide your own or we will add the cost of a new one ($1.00)
to your charges.
Examining Your Results:
The best way to view your sequence data is with the Applied Biosystems Sequencer Scanner
Software that is free and can be downloaded at the following link:
https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=600583
A brochure is also available (ABI Seq Scanner.pdf) which describes the features of Sequence Scanner.
This trace editor and viewer is a very useful tool that enables examination of raw data as well
as the text sequence and electropherograms. We encourage users to critically evaluate their raw
data for both successful and failed sequences.
Schedule:
The schedule for sequencing is posted on the GSC Calendar. Please
deliver your samples to 220 Morrill Science Building by 8:30 AM. If you have any
questions or problems, call the RIGSC manager at 874-5919.
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