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:

0. Make sure that you have an UNIX account to be able to receive your data. To be able to place sequencing orders, you need to inform SCF about your UNIX username, UniBi phone number, and the Kostenstelle you are allowed to use by reporting these information to SCF via the GFDB tasks interface
1. Supply high-quality DNA. Poor quality DNA is the most probable reason for the failure of the sequencing reaction. Although there are other (cheaper) ways to get pure DNA, we recommend Qiagen columns. "The Qiagen Guide to Template Purification and DNA Sequencing" gives many useful information. You can request this guide from Qiagen. An alternative protocol is shown here.
2. 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.
3. 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.
   • plasmid DNA: Supply at least 2 µg of your DNA at 250 ng per 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: To use the correct amount of template we need to know the size of your fragment. Supply primer-free fragments (we recommend Exo-SapIT cleanup from GE-Healthcare) and check the info-table for correct PCR fragment concentrations. Put the information in the respective field of the SeqOrderGFDB order form.
   • 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.
   • We also do small sequencing projects. We will completely sequence the fragment you give to us (in a vector - you supply the plasmid DNA as above). We only take fragments significantly larger than 1.4 kbp, i.e. those, which can not be sequenced completely with two reactions out of the vector (makes sense, doesn't it?). We order the primer and pay it - one reaction of such a sequencing project costs 18 €.
4. 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).
5. 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. Supply the primer at a concentration of 10 pmol per µl. Some standard primers don't need to be supplied since we have them in stock.
6. 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. See the SeqOrderGFDB FAQ for details.
7. 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!
   1. Place your sequencing request via SeqOrderGFDB. There are instructions in the forms - please read them.
8. When you deliver DNA and primer, you need to activate your order at the computer placed on top of the SCF refrigerator. You need to fill in the information about the location of your material in the freezer (Box No.). A request number will be automatically assigned to your request. Requests for which we do not have DNA or primer will not be processed. It is your responsibility to claim back your template DNA. We will not react to statements like "ask Lieschen Müller for the DNA", even if Lieschen Müller is working at SCF!.
9. The price for one sequencing reaction (i.e. one DNA with one primer, processed and loaded once) is 7 €. The person responsible for the given Kostenstelle will be informed, and the Kostenstelle will be debited by the "Buchhaltung" according to the number of reactions performed.
10. You will be informed by Email if there are new data.
11. Please make sure to remove 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. You can contact us via the GFDB tasks interface.