Frequently asked questions

  1. Why is my sequencing reaction not working (NNNNN)?

    When a sequencing reaction doesn't work at all, it's always difficult to determine the exact cause, but here are the most common problems :

    • Not enough DNA (quantify DNA and verify on gel).
    • Bad quality of DNA or presence of contaminants such as salts or ethanol (redo purification).
    • Not enough primer (redo dilution of primer at 5 µM or 5 pmol/µl).
    • Primer cannot bind, primer sequence is incorrect (check design and primer position).

    The protocol we use is not optimised for sequencing long repetitions or secondary structures (ex: hairpin). Sometimes, the sequencing goes very well, but for other cases we offer a special formula, where we modify certain parameters, such as adding DMSO. It's also possible to sequence BAC extremities. Contact us for more details.

  2. The concentration of my plasmid DNA is less then 200ng/µl, will the sequencing work anyway?

    In general, sequencing works just fine from 100 ng/µl (with the protocol we use).

    However, this is not an optimal concentration. The reliability of concentration and purity measures varies between spectros and between purification protocols. This is why we ask for a concentration of 200 et 300ng/µl, values where we observe best results. You can always validate your own personal method to determine which DNA concentration is enough for your sequencing.

    Please note that it is impossible for us to use a higher DNA volume in our reactions when the concentration is insufficient because the total reaction volume of our sequencing reaction is very limited (5µl).

  3. Do you sequence genomic DNA?

    No. You have to amplify your region of interest by PCR, and then purify and send the PCR product for sequencing.

  4. What primers do you provide?

    Primers we provide are T7+ (promoter), T7- (reverse), M13+, M13-, T3, SP6.

    T7+        5'- TAA TAC GAC TCA CTA TAG GG -3'
    T7- 5'- GCT AGT TAT TGC TCA GCG G -3'
    M13+ 5'- TGT AAA ACG ACG GCC AGT -3'
    M13- 5'- TCA CAC AGG AAA CAG CTA TGA C -3'
    T3 5'- ATT AAC CCT CAC TAA AGG GA -3'
    SP6 5'- GAT TTA GGT GAC ACT ATA G -3'


  5. When will my results be ready?

    From the moment we receive the samples, it usually takes two business days. You will receive an email when your results are available on the web site.

  6. Can you redo a sequencing reaction?

    If a sequencing reaction did not work and you are certain of your DNA concentration and purity, and of the presence of the primer site, contact us and we can redo some reactions.

  7. Do I need a PO number?

    A PO number is optional, but if you use one, it will be written on the bill sent each month. If necessary, please ask fo an open PO number to your administration and contact us for more details.

  8. Do I need to send my request form by email?

    Since sequencing requests are now inputted directly using our online form, you no longer need to send a request form by email.
    You MUST however attach a copy of the obtained PDF form when you deposit your samples at the platform. We need it to plan the sequencing and insure a fast and reliable service.
    If you don't attach your request form, your samples will not be processed.

  9. Do I need to purify my samples?

    Purification of you plasmid DNA is essential and a DNA sample that contain contaminants can be trouble for sequencing. PCR products have to be purified to clean out unincorporated primers and nucleotides.

Platform News

November 22, 2019
New!
We now offer a Single Cell sequencing service, using the chromium from 10X genomics.
contact us for more details at nextgen@iric.ca