SANGER Sequencing

Time Frame

We provide sequencing service from Monday to Friday. Results are available within 48 working hours. For high throughput, please contact us so that we may discuss the time required.

Automatic Sequencing Time Frame

Techniques

The platform use the Sanger technique to perform the sequencing. A polymerase chain reaction (PCR) is first conducted using Big Dye 3.1 terminators and thermocyclers in either 96 or 384 reaction-well microplate format. The sequence is then determined by capillary electrophoresis. All the samples are analyzed using one of Applied Biosystems platform, the ABI 3730xl Data Analyzer. This 96-capillary sequencer applies electrophoresis, fluorescence, and optical techniques to simultaneously analyze the sequence of 96 samples for more than 800 nucleotides. The ABI 3730xl can attains a capacity of more than 1,000 samples per day for a total of one million nucleotides. Through years of work with our numerous researchers, we have developed the experience and flexibility needed to carry out projects comprising of a few to several thousand sequences. It currently takes us 48 hours to analyze projects.

Open an account

To submit samples, you have to open a user account first. To do so, please contact by e-mail the lab manager, Patrick Laplante, and include the following information:

• The name of your researcher or your team;
• A password with 8 alphanumeric characters, starting by a letter;
• A phone number where you can be reached with the extension;
• An e-mail adress.

If your researcher or your team aren’t yet registered, an account will be necessary for them as well. The following information will be required:

• The first name and the last name of your researcher or the name of your team;
• Your department;
• Your address;
• The phone number where you can be reached;
• The payment method you want to use:
  • Credit Card
  • Purchase order
  • CHU budget number

Your user name will be sent to you by e-mail.

DNA Preparation

Please note that the better the quality of your DNA preparation are, the greater your chances of obtaining a good read length.

If you have a DNA with a high GC-content, a shRNA or a bisulfite DNA, please indicate it in the section Instruction Spéciale of the request form as well. GC-rich DNA sequences require distinct treatments to obtain a good sequence.

• PLASMID
  • Concentration: 50 ng/µl in Tris 10 mM pH 8,0 or H2O
    It is essential to correctly measure the concentration of your DNA. If the DNA is too concentrated, the sequencing reaction could drop off quickly and greatly diminish your read length. Conversely, an overly weak DNA concentration will produce excess background noise and introduce errors into the sequence analysis.
    It is best to avoid using EDTA when resuspending your DNA for the reason that EDTA inhibits the sequencing enzyme by chelating its co-factors
  • Volume: 15 µl per sample + 10 µl/reaction
    We ask for a minimum of 15 µl per sample. Then a surplus of 10 µl per sequencing reaction is necessary. If you provided less volume than that, then we will only be able to do one sequencing reaction without any possibility of resequencing or quantify on the Nanodrop 2000c.
  • DO 260/280 : between 1,8 and 2,0 et DO 260/230 : between 2,0 and 2,2
    These are good indicators of your DNA purity. RNA, solvents, salts and proteins could affect the quality of the sequencing.
  • Purification: Commercial columns
    We recommend using commercial purification kits to obtain quality DNA. The quality of DNA preparation directly affects sequencing results.
    Alkaline lysis extractions often leave contaminants that inhibit the sequencing reaction. When conducting a phenol/chloroform purification, be careful not to recuperate these organic solvents.
    Whatever method of purification is used, alleviate any traces of solvents, high quantities of salts, proteins, or RNA that will inhibit the sequencing reaction.
• PCR
  We dilute you PCR products according to their length. It is important to be precise about this information in order to obtain the right starting concentration before the sequencing reaction. When completing the order form, round the bp number up. It is equally essential that you only obtain one amplification product per PCR. Otherwise, you will have to separate them on an agarose gel before sending us your samples. However, agarose gel purification yields lesser sequencing results. Therefore, it would be preferable that you might adjust your PCR conditions to yield one amplicon per PCR. It’s also possible to sequence qPCR amplicons.
  • TO BE PURIFIED
    • Purification :
      • We provide, free of charge, PCR sample purification on fiber glass in order to eliminate oligos, dNTPs and salts.
        • Traces of PCR primers = double sequence
        • Traces of dNTPs or salts = shorter CRL and lower signal.
      • Volume : minimum of 20 uL and maximum of 50 uL per sample
        • If you have 1-4 reactions per sample : only one tube containing a minimum of 20 uL and a maximum of 50 uL
        • If you have more than 4 reactions per sample : for each multiple of 4 reactions, send a tube containing a minimum of 20 uL and a maximum of 50 uL
          • Exemple : if you have 11 reactions for one sample, it will be necessary to send 3 tube containing each a minimum of 20 uL and a maximum of 50 uL
      • Concentration : a unique and visible band on gel
  • ALREADY PURIFIED
    • Volume : minimum of 20 uL per sample
      • If you have 1-2 reactions per sample : minimum of 20 uL
      • If you have more than 2 reactions per sample : add 5 uL per additional reaction
    • Concentration: 5 ng/µl or more
      • If you dose with nanodrop before sending your PCR
        • Dilute the already purified PCR to 50 ng/uL
        • If the concentration is already around 50 ng/uL = do not dilute

Primers

• Specific primers
  • Concentration: 1,6 µM (~15-20 ng/µl)
  • Volume: 5 µl/reaction
  • Specifications :
    For each sequencing reaction, we require that the primer length is between 18 and 24 bases with a GC content of more than 50%.
    Your primer must be specific to your DNA templates in order to avoid double sequencing.
    Please avoid errors at the 3-prime end of the oligos, as they make the sequencing reaction impossible.
    Likewise, please avoid secondary structures in the primer: the sequencing reaction will be affected.
• Universal primers
  

  Nom	Séquence	Tm (°C)
  SP6	TATTTAGGTGACACTATAG	42
  T3P	CGAAATTAACCCTCACTAAAGG	51
  T5-F	AATTTATTTGCTTTGTGAGC	47
  T5P	CCCGAAAAGTGCCACCTG	57
  T7-EEV	AAGGCTAGAGTACTTAATACGA	50
  T7p	TAATACGACTCACTATAGG	45
  T7t	GCTAGTTATTGCTCAGCGG	53
  T7p-1G	TAATACGACTCACTATAG	41
  T7-3G	TAATACGACTCACTATAGGG	48
  BGH	TAGAAGGCACAGTCGAGG	54
  M13F-20	GTAAAACGACGGCCAGT	53
  M13F-47	CGCCAGGGTTTTCCCAGTCACGAC	64
  M13R-17	CAGGAAACAGCTATGAC	47
  M13R-20	GCGGATAACAATTTCACACAGG	55
  M13R-48	AGCGGATAACAATTTCACACAGGA	57
  3-AD	AGATGGTGCACGATGCACAG	58
  3-DNABD	TTTTCGTTTTAAAACCTAAGAGTC	49
  Ac5	ACACAAAGCCGCTCCATCAG	58
  Attr2_rev	AGGTTCCTTCACAAAGATCC	52
  Amp-R	ATAATACCGCGCCACATAGC	56
  5-AOX1	GACTGGTTCCAATTGACAAGC	54
  3-AOX1	GCAAATGGCATTCTGACATCC	55
  CYC1	GCGTGAATGTAAGCGTGAC	54
  CAG-F	TCGGCTTCTGGCGTGTGACC	63
  CAG-R	TGTCCCCATAATTTTTGGCAGAGG	58
  C-CMV-24	TATTAGGACAAGGCTGGTGGGCAC	61
  CMV-Forward	CGCAAATGGGCGGTAGGCGTG	64
  N-CMV-30	AATGTCGTAATAACCCCGCCCCGTTGACGC	67
  CPBP-FW	GCAATCAGGAAGTCGGTACTAA	54
  CPBP-RW	ATTTGCGCCCATGCTTTG	55
  DsRed1-C	AGCTGGACATCACCTCCCACAACG	63
  EXFP-R	GTCTTGTAGTTGCCGTCGTC	56
  EBVrev	GTGGTTTGTCCAAACTCATC	52
  EF1a_fwd	TCAAGCCTCAGACAGTGGTTC	57
  EF1a_Rev	CCCAACTTCTCGGGGACTGTG	60
  EGFPC2_fwd	CCCAACGAGAAGCGCGATC	59
  EGFPC2_rev	CACTCAACCCTATCTCGGTC	55
  EGFP-Cfor	AGCACCCAGTCCGCCCTGAGC	67
  EGFP-Nrev	CGTCGCCGTCCAGCTC	59
  GFP-Cfor	GGTCCTTCTTGAGTTTGTAAC	51
  GFP-Fw	GAAGCAGCACGACTTCTTC	54
  GFP-reverse	GGGTAAGCTTTCCGTATGTAGC	55
  pEGFP-C	CCTGCGGAGTTCG	49
  pEGFP-C-3	TATGGCTGATTATGATCAGT	48
  pEGFP-C-5	CATGGTCCTGCTGGAGTTCGTG	60
  pEGFP-N-3	CGTCGCCGTCCAGCTCGACCAG	66
  pEGFP-N-5	TGGGAGGTCTATATAAGCAGAG	53
  GLprimer2	CTTTATGTTTTTGGCGTCTTCCA	54
  GLprimer4	GGGTAGAATGGCGCTGGG	68
  RVprimer3	CTAGCAAAATAGGCTGTCCC	53
  RVprimer4	GACGATAGTCATGCCCCGCG	61
  H1F	TGTTCTGGGAAATCACCATA	51
  HA-reverse	GCGTAGTCTGGGACGTCGTATGGGTA	63
  Ha-fw-long	TACCCATACGATGTTCCAGATTACGCT	59
  hEF1aprom-F	AACTGGGAAAGTGATGTCGTG	55
  hGata4-rev	ATTGTGGATGAATACTGCC	50
  hGH-pA-R	CCAGCTTGGTTCCCAATAGA	54
  hU6-F	GAGGGCCTATTTCCCATGATT	54
  IRES-F	TGGCTCTCCTCAAGCGTATT	56
  IRES-R	CCTCACATTGCCAAAAGACG	55
  Lambda-t0	GGTCATTACTGGAGTCTTG	50
  LKO1-5	GACTATCATATGCTTACCGT	50
  LucNrev	CCTTATGCAGTTGCTCTCC	47
  Luc-F	AGTCAAGTAACAACCGCGA	54
  Rluc-fwd	GCCAGGAGGACGCCCCCG	67
  RlucII-fv2	GGCCGGATATAGAGGAGGATATT	56
  Rluc-Rev	TCGCTGTCGTAGTAGTTGATGA	55
  MBP-F	GATGAAGCCCTGAAAGACGCGCAG	62
  MT_Forward	CATCTCAGTGCAACTAAA	47
  mCherry-F	CCCCGTAATGCAGAAGAAGA	55
  mCherry-R	TTGGTCACCTTCAGCTTGG	55
  mCherry-CTer	CCCACAACGAGGACTACA	54
  Neo-F	CGTTGGCTACCCGTGATATT	55
  Neo-R	GCCCAGTCATAGCCGAATAG	55
  pASK-fwd	GAGTTATTTTACCACTCCCT	49
  pASK-rev	CGCAGTAGCGGTAAACG	54
  pBR322ori-F	GGGAAACGCCTGGTATCTTT	55
  F1ori-F	GTGGACTCTTGTTCCAAACTGG	56
  F1ori-R	AGGGAAGAAAGCGAAAGGAG	55
  pBABE3	CCCTAACTGACACACATTCC	53
  pCasper-hs	GCAACTACTGAAATCTGCCAAG	54
  pcDNA3-4-REV	CAACATAGTTAAGAATACCAGTC	49
  pENTR-F	CTACAAACTCTTCCTGTTAGTTAG	51
  pENTR-R	ATGGCTCATAACACCCCTTG	55
  pGEXfw	GGCAAGCCACGTTTGGTG	58
  pGEXrv	GAGCTGCATGTGTCAGAGG	56
  pGEXfw-long	GGGCTGGCAAGCCACGTTTGGTG	66
  pGEXrv-long	CCGGGAGCTGCATGTGTCAGAGG	64
  pGK-forward	CATTCTGCACGCTTCAAAAG	53
  pJETF	CGACTCACTATAGGGAGAGCGGC	61
  pJETR	AAGAACATCGATTTTCCATGGCAG	56
  pLightSwitch-F	TCCATCAAAACAAAACGAAACAA	52
  pLightSwitch-R	AGTCGAGCACGTTCATCTGCTT	59
  pMSCV	GAGACGTGCTACTTCCATTTGTC	56
  pMSCV-5'	CCCTTGAACCTCCTCGTTCGACC	61
  Polyhedrin	AAATGATAACCATCTCGC	47
  Poly-T	TTTTTTTTTTTTTTTTTTTTTTTT(A/C/G)	42
  PQE-Rev	GTTCTGAGGTCATTACTGG	50
  pTRIPZ-F	GGAAAGAATCAAGGAGG	47
  pTRIPZ-R	TCTGACGTGGCAGCGCTCGCC	67
  p-vitro-fwd	TTTTGAGCGGAGCTAATTCTCGGG	59
  RPC5-SR	ATGCCTGCTATTGTCTTCC	52
  RSV-fwd	CATGCCGATTGGTGGAAGTAAG	56
  S-tag	GAACGCCAGCACATGGACA	58
  SK	TCTAGAACTAGTGGATC	43
  SV40pA-R	GAAATTTGTGATGCTATTGC	48
  SV40pro-F	TATTTATGCAGAGGCCGAGG	54
  SV40rev	GGTATGGCTGATTATGATC	47
  SV40-pA-JH	AAAAAACCTCCCACACCTCC	55
  Tet-pLKO-neo-F	GGCAGGGATATTCACCATTATCGTTTCAGA	60
  TypeIII-IV	CGGATAACAATTTCACACAG	49
  V5_C-term	ACCGAGGAGAGGGTTAGGGAT	59
  WDR36exon18-F	GATGGCATCTATGACATAAGTC	51
  WDR36exon19-R	GCATTGTCAGTGCTGTCTTAC	54
  WPRE-R	CATAGCGTAAAAGGAGCAACA	53
  rb_glob_PA	CCCATATGTCCTTCCGAGTG	55
  Bglob-pA-R	TTTTGGCAGAGGGAAAAAGA	53
  Puro-R	GTGGGCTTGTACTCGGTCAT	57
  LNCX	AGCTCGTTTAGTGAACCGTCAGATC	59
  pUast-Attbf	CTGCAGTAAAGTGCAAGTTAAAGTG	55
  pUast-Attbr	CATCAGTTCCATAGGTTGGAATCTA	54
  SSU27	AGAGTTTGATCMTGGCTCAG	54
  SSU534R	ATTACCGCGGCTGCTGG	59
  16S-27F	AGAGTTTGATCCTGGCTCAG	54
  16S-27F(M)	AGAGTTTGATCMTGGCTCAG	54
  16S-534R	ATTACCGCGGCTGCTGG	59
  16S-1541R	AAGGAGGTGATCCAGCCGCA	61
  18S-NS1	GTAGTCATATGCTTGTCTC	47
  18S-NS4	CTTCCGTCAATTCCTTTAAG	49
  18S-NS8	TCCGCAGGTTCACCTACGGA	61
  ITS1	TCCGTAGGTGAACCTGCGG	60
  ITS4	TCCTCCGCTTATTGATATGC	52

Shipping’s rules

• All samples must be shipped in 1.5 ml Eppendorf microtubes, in PCR strips tubes or in 96-well microplates.
• Always write your order number on the top of your tubes. Your order number will be inscribed on your order confirmation page.
• Write the names of your samples and primers legibly on the side of your tubes. If the names of your samples are long, you can identified them with the reference well ID in the submission form (ex: A1, B1, C1, etc) or numbered them (ex: 1, 2, 3, etc)
• If Paraffin is used to close your tubes, please apply a small quantity.
• Each tube must have a minimum of 10 µl.
• Please do not use tape to identify your tubes. It has the unfortunate habit of falling off at -20ºC.
• Please do not use tape to join your samples together. It then becomes difficult to separate them and it remove the ink on your tubes. Rather, use an envelope, a storage bag (ex: Ziploc®) or other easy-to-handle container.
• It is not necessary to put your DNA on ice or dry ice if they are not contaminated by endonucleases.
• It is recommended to protect your samples during transport: use a bubble wrap envelope or a solid container like a Falcon tube of 50 mL.

Please ensure that your samples do not arrive here on the weekends or holidays.

Adresse d’envoi

• External clients:

Patrick Laplante
SANGER Sequencing Platform, CRCHU de Québec – Université Laval
2705, boulevard Laurier, T0-72
Quebec City, Quebec
CANADA G1V 4G2

• Internal clients:
  • Your samples can be left at the reception desk of bloc T in Room TR-72 or in the entrance of the Infectious Disease Research Center in Room RC-709
  • You can bring them to room R-4756 if you are authorized to do so.
  • You can send them to us by internal courier; however, you must allow for one extra day for delivery.