Sanger Sequencing Service

In order to submit samples, you must first open a user account. To do so, please email the platform manager, Patrick Laplante, including the following information:

• The name of your researcher or team;
• An 8-character alphanumeric password starting with a letter;
• The phone number to reach you along with the extension;
• An email address.

If your researcher or team is not already registered, you will also need to open an account for them. The following information will then be required:

• The researcher’s full name or the team name;
• Your department;
• Your address;
• The phone number;
• The payment method you will use:
• Credit card
• Purchase order
• CHU budget number

Your username will be sent to you by email afterward.

The better the quality of your DNA preparation, the higher the chances of obtaining a good read length.
If your DNA is GC-rich, consists of shRNA, or has been bisulfite-treated, please mention it in the Special Instructions section of the order form (write in this section: GC-rich sample).

The secondary structures they contain can affect the quality of the sequence. Therefore, we use alternative protocols.

PLASMIDE 

• Concentration: 50 ng/µL in 10 mM Tris pH 8.0 or H2O
• It is important to accurately measure the concentration of your DNA. If too concentrated, the sequencing reaction may quickly drop off, greatly reducing the read length. Conversely, too low DNA concentration would result in high background noise and introduce errors in determining your sequence. Be careful when resuspending your DNA as EDTA inhibits the sequencing enzyme by chelating its cofactors
• Ratio: OD 260/280: between 1.8 and 2.0 and OD 260/230: between 2.0 and 2.2
• These are good indicators of the purity of your DNA. If there is residual RNA, solvent, salts, or proteins in your sample, the sequencing quality will be affected.
• Volume: 15 µL per sample + 10 µL/reaction
• We require a minimum of 15 µL per sample. Additionally, a surplus of 10 µL per sequencing reaction is necessary. If you cannot provide the requested volume, we can only perform one sequencing reaction without the possibility of redoing or verifying the dosage on the Nanodrop 2000c.
• Purification: commercial column type
  It is recommended to use a commercial purification kit to obtain high-quality DNA. The better prepared your DNA is, the higher the sequencing quality will be. Be cautious with alkaline lysis extractions: there are often contaminants that inhibit the sequencing reaction. If you perform a phenol/chloroform purification, take care not to recover these organic solvents. Regardless of the type of purification used, it should not leave traces of solvent, or high amounts of salts, proteins, or RNA that would inhibit the sequencing reaction. Therefore, we strongly advise a second wash during miniprep-style purifications and vacuum centrifugation before elution to remove any trace of contaminants on the column.

PCR

We dilute your PCR products based on their base pair length. It is important to be precise with this information to obtain the correct starting concentration before the sequencing reaction. When filling out the order form, round the number of base pairs to the nearest higher number. Additionally, it is important to have only one amplification product per PCR, otherwise you will need to separate them on agarose gel before sending us your sample. However, the purified fragments may not always yield good sequencing results. Therefore, it may be preferable to adjust your PCR conditions to produce a single product. It is also possible to sequence qPCR amplicons.

TO BE PURIFIED

• Purification :
• We offer complimentary purification of PCR samples on glass fiber membrane to eliminate oligos, dNTPs, and salts.
  • PCR primer residues = double sequencing.
  • dNTP or salt residues = decreased CRL length and signal intensity
• Volume: minimum 20 µL and maximum 50 µL per sample.
  • If you have 1 to 4 reactions per sample: one tube containing minimum 20 µL and maximum 50 µL.
  • If you have more than 4 reactions per sample: for each multiple of 4 reactions requested, send one tube containing minimum 20 µL and maximum 50 µL.
    • Example: if you have 11 reactions for one sample: you will need to send us 3 tubes, each containing at least 20 µL and at most 50 µL of this sample.
• Concentration: a single, visible band on gel.
  • A PCR with good efficiency will easily yield this result.

ALREADY PURIFIED

Volume: minimum of 20 µL per sample.

• If you have 1 to 2 reactions per sample: minimum of 20 µL.
• If you have more than 2 reactions per sample: add 5 µL per additional reaction.

Concentration : ≥ 5 ng/µL

• A PCR with good efficiency will easily yield more than 5 ng/µL. If you cannot achieve such yields, it may affect the sequencing reaction.
• If you measure with Nanodrop before sending your PCR:
• Dilute the already purified PCR to 50 ng/µL.
• If the measurement is already around 50 ng/µL or lower: do not dilute your samples.

Specific Primers

• Concentration : 1,6 uM (approximately 15 to 20 ng/µl)
• Volume : 5 µL/reaction
• Specifications: We require one primer per sequencing reaction that resembles those used for PCR. For example, an 18 to 24 mer with a GC content of over 50% is adequate. Your primer must also be specific to your DNA template to avoid double sequences. Be cautious of errors in the 3′ end of the primer, as they render the sequencing reaction impossible. Avoid secondary structures in your primers, as they will affect the sequencing reaction yield.

If your universal primer is not on this list, we’ll be happy to have it synthesized and added.

List of universal primers (In French)

We offer sequencing services from Monday to Friday, and results are available within the next 48 business hours. For very high-throughput projects, please call us to discuss a timeline.

• All samples must be sent to us in 1.5 mL microtubes (Eppendorf style), PCR tubes in strips, or in 96-well microplates. When you have multiple samples (24 or more), it’s easier for us to handle plates or strips. Additionally, you’ll have less labeling to do, reducing handling errors.
• Always write your order number on the top of your tubes; it will be provided on the order confirmation page

• Clearly write your sample and primer names on the side of your tubes. If your sample names are very long, you can also identify your tubes with the corresponding well numbers from the order form. (e.g., 1, 2, 3,… or A1-B1, C1-D1,…)

• If you use parafilm to seal your tubes, use the minimal amount.
• Each tube must have a minimum of 10 µL.
• Do not use gummed tape to identify your tubes, as it tends to peel off at -20 ºC.
• Do not use gummed tape to bind your samples together; it’s difficult to separate and erases ink on the tubes. Instead, use an envelope, a packaging bag (e.g., Ziploc®), or any other easy-to-handle container.
• It is not necessary to put your DNA on ice or dry ice if it is not contaminated by endonucleases.
• It is recommended to protect your samples during transport by mail or courier: envelope with bubble wrap or in a sturdy container like a 50 mL Falcon tube.

Caution: Make sure your samples do not arrive during the weekend or on a holiday.

For external senders:

• Patrick Laplante
  Plateforme de séquençage et de génotypage (CRCHU de Québec-CHUL)
  2705, boulevard Laurier, T0-72
  Québec, Québec
  CANADA G1V 4G2

For internal senders:

Your samples can be left at the reception of block T at room TR-72 or at the entrance of the Centre de recherche en infectiologie at room RC-709;

You can bring them to us at room R-4756 if you have access;

You can send them to us through internal mail; however, please allow an additional day.