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Plate-forme d'Analyses Génomiques de l'Université Laval

Sample preparation

  1. General instructions for DNA preparation
    • DNA sequencing requires careful preparation of templates and primers. The following instructions should be followed before submitting your samples. It is of utmost importance that you follow these instructions to the letter in order to ensure the success of your sequencing reactions.
    • Template DNA and primers must be prepared so as to respect high standards of quality and quantity.
    • The quality of the template DNA is the main factor ensuring the success of sequencing. Poorly prepared samples containing an unacceptable quantity of salts, proteins, RNA or other contaminants will inevitably lead to undesirable sequencing results.
    • The DNA samples must be in nanopure water or buffer solution (10 mm Tris-HCl pH 8.0). Never use TE buffer solution, as the EDTA can inhibit the reactions.
    • For the preparation of the plasmids, we suggest the following bacterial strains: DH5 alpha, HB101 or XL-1 Blue.
      We do not recommend JM101 and other strains of the same series.
    • Sequencing reactions are performed with universal or specific (designed by the client) primers
  2. Suggested protocols

  3. Preparation of the samples for forwarding
  4. Universal and specific primers
    • Universal primers
    • List of the most frequently used universal primers available at our service without any additional costs:
      Name 5'-3' Sequence Tm
      M13 Reverse (-20)1 CAG GAA ACA GCT ATG AC 48.5ºC
      M13 Reverse (-48)1 AGC GGA TAA CAA TTT CAC ACA GGA 69.4ºC
      M13 Forward(-21)1 TGT AAA ACG ACG GCC AGT 59.8ºC
      M13 Forward ( -47)1 CGC CAG GGT TTT CCC AGT CAC GAC 79.0ºC
      T3 promoter CGA AAT TAA CCC TCA CTA AAG G 62.5ºC
      T7 promotor2 TAA TAC GAC TCA CTA TAG GG 51.4ºC
      T7 terminator GCT AGT TAT TGC TCA GCG G 60.5ºC
      SP6 promoter TAT TTA GGT GAC ACT ATA G 43.5ºC
      (T)24(A/C/G)3 TTT TTT TTT TTT TTT TTT TTT TTT (A/C/G) 53.5ºC
      1In the case of sequencing with M13 forward and M13 reverse, it is mandatory to specify their positions. If the positions are not indicated, we will use M13 Reverse(-48) and M13 Forward(-47) by default.
      2The pCI and pSI vectors from the Promega company have a different T7 promoter primer than the one we provide. The last 3' base is different between the two primers.
      3For cDNA clone sequencing towards the polyA(>18As) tail.
       
    • Specific primers
    • These primers must be sent by the client. Please follow these instructions:
      • Make sure that the concentration is 1.5 µM (5µl per reaction).
      • The primers for sequencing of BACs or YACs must be provided at a concentration of 10µM (5µl per reaction).

    • Helpful hints for the design of specific primers
    • There are many application softwares that enable specific primer design. The base rules for obtaining efficient primers for sequencing are:
      • good purity level : > 95% full length
      • Length: 18-24 bases with a GC-content above 50%
      • Melting temperature (Tm) between 50°Cand 60°C
      • No secondary structure
      • No alternative binding sites on the DNA
      • No mismatches
      • Avoid repetition of the same base (4 and more)