DNA sequencing information

The Sequencing Core Facility unit is located in G1 (lab building used by the CeBiTec). If you would like to have DNA sequenced by SCF, please observe the following:

    1. Make sure that you have a Cebitec account to be able to receive your data. To be able to place sequencing orders, you need to inform SCF about your Cebitec username, phone number, and the Kostenstelle/PSP-Element you are allowed to use by reporting these information to SCF using the Task Interface.
    2. Supply high-quality DNA. Poor quality DNA is the most probable reason for the failure of the sequencing reaction.
    3. Supply the DNA in 10 mM Tris pH 8.0 but not in TE! EDTA interferes even at lowest concentration with the cycle-sequencing reaction and leads to NO results. If you need to precipitate your DNA, we highly recommend to perform a NH4Ac-precipitation: 1 vol DNA solution, 1 vol 4M NH4Ac, 6 vol EtOH; mix and centrifuge at RT. This precipitation reduces contaminating nucleotides which interfere with OD determination, as well as contaminating DNA or RNA oligonucleotides which can cause random priming in the sequencing reaction.
      1. Some bacterial stains are better suited for preparing template DNA than others:
          E.coli strain designation comment
        good strains: DH1, DH5alpha, C600, HB101 please use these where ever possible
        acceptable strains: JM109, XL-1blue RNAse treatment required!
        strains NOT to use: MV1190, other JMxyz-strains, etc. nuclease activity, base modifications

        In general, the cells should be endA-, and should also be also defect in a number of base modification systems.
    4. We can sequence "normal" (ds/ccc) plasmid DNA, clean PCR fragments, cosmid DNA, and BACs. There are still problems with lambda DNA; this DNA is usually not sufficiently clean (although we did successfully sequence lambda DNA labelled as "cosmid" ). You may consider to PCR amplify the insert and use this PCR fragment as template. Please note: It is not possible to sequence genomic DNA by Sanger.
      • plasmid DNA:
      • Per reaction supply at least 6 ul of DNA at a maximum concentration of 250 ng/µl. Deliver sufficient amounts to allow safe pipetting and inform us about the vector used and the size of the construct (if > 8 kbp).
      • PCR fragments:
      • Supply primer-free single fragments (I strongly recommend Exo-SapIT cleanup from GE-Healthcare, or Exo-CIP Rapid PCR Cleanup from NEB), performed in a cycler.
      • Please: Do not use any spin columns for a proper single band PCR product, just supply the enzymatic cleaned PCR-products for sequencing!
      •  For gel-cut fragments you have to use the protocol you are used to and which works best in your hands. Adjust the concentration to 1/10 of the length of the expected PCR product length (e.g. for a 500 bp fragment the concentration should be 50 ng/ul)
      • cosmid DNA:
      • We need at least 2 µg of very clean cosmid DNA per reaction. We recommend Qiagen columns for purification (there is a special protocol for BAC DNA). Supply your cosmid DNA at 1 µg per µl.
      • BAC DNA:
      • We need at least 1 µg of very clean BAC DNA per reaction. We recommend Qiagen column purification for template preparation. Supply your BAC DNA at 1 µg per µl. 
    5. Since the correct DNA amount is really important for good sequencing results, please try to be as exact as possible. Incorrect DNA concentrations lead to NO results (too low: signals too weak; to high: reduced accuracy, similar to an overexposed autoradiography).
    6. Make sure that your primer anneals at 55 °C or higher. Since the cycle-sequencing reaction is a linear PCR reaction, there will be NO results with primers that do not anneal at 55 °C. We recommend primers with 20 to 22 nt's and about 50% GC.
    7. Supply your custom primer at a concentration of 10 pmol per µl and provide 10 ul for a single reaction. For several reactions provide 10 ul more primer than the number of reactions you request, e.g.: you want 3 reactions to be done with the same primer supply 13 ul primer in total. 
    8. Make sure to give the correct Kostenstelle which should be debited - SeqOrderGFDB only allows you to use Kostenstellen you are registered for. Your order (which is generated by SeqOrderGFDB) must be signed by the person responsible ("zeichnungsberechtigt") for the given Kostenstelle. You can also become authorized to place sequencing orders without your group leader's signature. 
    9. Use short abbreviations for your DNA and Primer: a maximum of 4 characters for each DNA and primer is possible. Special characters like #, ", ', ´, etc are not accepted. Your username will be added by default to the final data file - no need for initials. The template & primer designations are used to name the resulting data file. Please label the tubes containing your material clearly on the lid. MAKE SURE THAT WE CAN READ THE LABELLING OF THE TUBES!
    10. When you deliver DNA and primer, you need to put your samples, primer and ordersheet into the mailbox placed next to the entrance of the sequencing unit in G1, or into the mailbox outside on the right side of the Cebitec entrance.  A request-number will be automatically assigned to your request when we start working on your samples. Requests for which we do not have DNA or primer will not be processed. It is your responsibility to claim back your template DNA. 
    11. Please make sure to take back your DNA and primer from the "DNA OUT" box after you got your results. Samples older than three weeks will be thrown away.
    12. Please report problems directly to us. We can only improve if we get feedback from you - ... positive feedback is also welcome.