SUBMISSION GUIDELINES

Submission Overview

Step 1: Prepare samples specifically for the preferred service level

Step 2: Enter your order request only using dnaLIMS, and submit samples with a printed copy of your request form together in a sealed bag.

Step 3: Drop off in-person in the refrigerated dropbox on the main floor of the Pharmaceutical Sciences building (contact us for details), by campus mail, or by courier to:

Sequencing and Bioinformatics Consortium
University of British Columbia
Room 3124 - PharmSci Building
2405 Wesbrook Mall
Vancouver, BC V6T 1Z3

Submission Deadlines:

9:00 am for SBC_Core-Prepared samples
2:00 pm for all other sample types

Submission Guidelines

Following these guidelines will ensure that your samples are processed without delay.

Preparing Your Order

A maximum of 92 samples can be submitted in each order to allow for the inclusion of controls. Your order request must be submitted online using dnaLIMS prior to physically submitting your samples:

Orders ≤ 48 samples: submit in 8-strip PCR tubes
- Label the side of the tubes with the sample number
Orders > 48 samples: submit in 96-well PCR plate
- Submissions in 96-well plates must be in the following orientation:

Note: We can provide you with strips or plates if needed, please contact us at sanger.submissions@ubc.ca for details.

The SBC offers four service levels for Sanger sequencing, each with its own submission guidelines:

Template and Primer Requirements

**Important: Templates and primers must be resuspended in molecular biology grade water or 10 mM Tris pH 7-8 with no EDTA, as EDTA will interfere with the ion concentration in the sequencing reaction.

Template Considerations

Of primary importance to the success of DNA sequencing reactions is the quality and quantity of sample provided. SBC cannot be held responsible for failures in sequencing which are related to poor preparation of the DNA template used, or failure to follow recommended guidelines.

Contaminated template will give high background noise and poor, or no, sequence information
Many of the commercially-available extraction and purification kits (eg. Qiagen, Promega, Wizard Plus, Sigma) are recommended
It is critical that the concentrations are measured carefully with UV absorbance / Nanodrop quantification (not fluorometric), as incorrect quantitation (whether higher or lower) will result in poor sequencing results.
Modified conditions are available for “difficult” templates (GC-rich, poly-T)

Primer Considerations

Recommendations when designing custom sequencing primers:

Tm between 55°C and 60°C
Formula: ºC = 4(G+C) + 2(A+T)
“G” or “C” at 3’end
Avoid primers with runs of 3 or more of the same base
Use primer design software to avoid primers with hairpin or dimer characteristics

Standard Sequencing Primers available at SBC

Standard Templates	Minimum Quantity Required (per reaction)	MInimum Quantity Required (per submission)	Concentration	Volume
plasmid DNA	200 ng	400 ng	40 - 250 ng/µL	>10 µL per reaction
purified PCR product (100 - 300 bp)	5 ng	10 ng	1 - 20 ng/µL	>10 µL per reaction
purified PCR product (301 - 1,000 bp)	15 ng	30 ng	3 - 40 ng/µL	>10 µL per reaction
purified PCR product (over 1,000 bp)	50 ng	100 ng	10 - 200 ng/µL	>10 µL per reaction

Other Templates
cosmid/fosmid/BAC (over 20 kbp)	600 ng	1200 ng	500 - 800 ng/µL	>10 µL per reaction

Custom primer, if required*
primer, submitted separately from template			5 pmol/µL	>5 µL per reaction

Standard Template	Final Amount Required	Total volume
Plasmid DNA	400 ng	14 µL
purified PCR product (100 - 300 bp)	10 ng	14 µL
purified PCR product (301 - 1,000 bp)	30 ng	14 µL
purified PCR product (over 1,000 bp)	100 ng	14 µL
primer	10 pmol	included in above

Other Templates
cosmid/fosmid/BAC (over 20 kbp)	1200 ng	14 µL
primer	10 pmol	included in above

Template	200 ng
Primer	5 pmol
BigDye® dilution mix	3 µL
dH₂O to final volume:	10 µL

Template (100-300bp)	5 ng
OR (301-1000bp)	15 ng
OR (over 1000bp)	50 ng
Primer	5 pmol
BigDye® dilution mix	3 µL
dH₂O to final volume:	10 µL

Template	600 ng
Primer	5 pmol
BigDye® dilution mix	3 µL
dH2O to final volume:	10 µL

SUBMISSION GUIDELINES

Submission Overview

Submission Deadlines:

Submission Guidelines

Preparing Your Order

The SBC offers four service levels for Sanger sequencing, each with its own submission guidelines:

A: SBC_Core-Prepared

Template and primer are submitted separately. Our staff will prepare the reaction and add sequencing chemistry, perform cycling, post-reaction clean-up, and electrophoreses.

B: User-Prepared, Pre-Mixed

Template and primer are submitted pre-mixed with water. Our staff add sequencing chemistry, perform cycling, post-reaction clean-up, and electrophoreses.

C: User-Prepared, SBC_Core-Cleaned

User performs the cycle sequencing with ABI Big Dye 3.1 Cycle Sequencing kit, and submits cycled product for post-reaction cleaning, injection, and electrophoretic separation.

D: User-Prepared, User-Cleaned

User performs the cycle sequencing and post-reaction clean-up, and submits samples for injection and electrophoretic separation.

Protocol: Standard Sequencing (Plasmid, PCR Product)

Reaction Set-up:

Cycling Protocol:

Protocol: Specialty Sequencing (COSMID, BAC)

Reaction Set-up:

Cycling Protocol:

Template and Primer Requirements

Template Considerations

Primer Considerations

Sequencing + Bioinformatics Consortium, Pharmaceutical Sciences Building

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