none
 CSUPERB
          MicroChemical Core Facility
Home Service Technical Support Price Info & Order Map Direction Links & Software Contact + People


We provide the following high quality and fast services to our customers.

  1. DNA Sequencing
    1. Self-Service sequencing
    2. Full-service sequencing
  2. Genotyping
  3. Templiphi: direct sequencing from bacteria glycerol stock
  4. 96-well plasmid extraction and purification
  5. Purification of PCR product for sequencing

  1. DNA Sequencing:
    We pick up samples from NLS 316 every day at 9:00 am. Samples are sequenced in the order they are received. Deposit your samples on the top shelf of the freezer and leave the order form in the top basket by the back door.
    1.   Self-Service sequencing:
      • We provide sequencing from templates such as plasmids, PCR products, lambda phage, cosmids, small viral genomes and BACs.
      • Submit one tube containing BOTH your template and primer for each separate reaction, in the following quantities. These amounts and ratios are important. Your DNA must be ultrapure! See template preparation.
      • Preferred submissions are in 96 well plates, 8-strip type PCR tubes with caps, or individual 0.2 ml PCR tubes, to assist multichannel pipetting. Individual 0.5 and 1.5 ml. microfuge tubes are also accepted.
      • We also provide a set of standard primers such as T3, T7 and SP6, free of charge.

          Quantity
          Plasmid PCR Product
        Template ~1-2 ug 20ng/100bp
        Primer*a 10 pmols per ug DNA 6.4 picomoles/50ng
        UltraPure H2O varied varied
        Total*b 12 uL 12 uL

        *a: If you want us to add one of our stock primers, mark the form "please add primer". For a list of available primers, see (link to available primer page). Stock primer is provided free of charge. Self service reactions, where we add stock primer, will only be charged at the self service rate.
        *b: Label tube and order sheet "SS" to indicate that this is a Self-Service Reaction.

      • Primer Design Considerations
        • Good sequencing primers are generally 18-20 bases long, have around 50% GC content and Tm's between 55 and 75 degrees. Use of an oligo design computer program can greatly simplify designing primers.

      IMPORTANT

      In order for us to better serve your sequencing needs, as well as to help us troubleshoot all the sequencing related problems, we require a Gel Image to be provided for each sample to be run. In case you have a large amount of samples, please show us a few representatives. A copy of the image from your lab notebook is fine. The image can be attached with the order form, or sent via e-mail.
      Thank you very much for your cooperation!

    2.   Full-service sequencing
      • You provide purified DNA template and primer in separate tubes.
      • We will check the concentration of the DNA you provided, assemble the reactions, add the fluorescent dye mix, and run it on a gel.
      • If data is good after the first run, you are charged $15. If it is not good, we will adjust reaction conditions and run the reaction again a second time for no extra charge.


  2. Genotyping:
    • We are pleased to offer genotyping services. We have both ABI 3100 and ABI 310 genetic analyzer which can take samples in 96 well format.
    • We provide the following dye sets, DS-30 and DS-31, which use ROX as size standard, and DS-33, which uses LIZ as size standard. In addition, we offer two special size standards, the ROX1000 and LIZ600, to accommodate the needs of analyzing fragements which extend the standard sizes.
    • We also provide training and consultant service in data analysis using GeneMapper® v4.0. This service may be subject to a fee.
  3. Templiphi: direct sequencing from bacteria glycerol stock
    • We offer plasmid DNA amplification directly from your bacterial glycerol stock, so you no longer need to isolate plasmid DNA for sequencing. We use the TempliPhiTM DNA Amplification kit, sold by GE Biosciences (formerly Amersham). Starting from picogram amounts of plasmid, TempliPhi generates microgram quantities of template DNA utilizing bacteriophage Phi29 DNA polymerase and rolling circle amplification. See PDF file.

    • Sample requirements:
      10 ul of glycerol stock culture. Stocks must be viable and delivered directly to our lab.
  4. 96-well plasmid extraction and purification:
    • We also offer an alternative to TempliPhi: plasmid purification in 96 well format, from your glycerol stock cultures. The stocks are used to inoculate 96 well block cultures that are grown overnight, then treated with chloramphenicol to amplify plasmid DNA. The cells are centrifuged and pellets harvested, then processed with a 96 well filterplate method based on alkaline lysis, yielding purified plasmid DNA that can be used in sequencing reactions.

    • Sample requirements:
      10 ul of glycerol stock culture in 96 well plate. Stocks must be viable and delivered directly to our lab.
    • Important:
      Cultures have to be in a 96-well plate when they are delivered to us. There will be an extra charge for colony picking from plates.
    • Purification of PCR product for sequencing
      • We offer PCR purification of individual samples or 96 well plates, using a Millipore filterplate protocol. The minimum volume of PCR reaction is 25 ul. Check your PCR reactions on a gel for product before submitting them.

      • Special attention for ExoSap: samples containing PCR product purified with ExoSap must be kept frozen after addition of the primer to template. Please hand carry your samples to our lab on ice and put them in our -20oC freezer. The reason for special handling is that the Exo I component of ExoSap sometimes remains active even after the deactivation step, and will destroy the sequencing primer, yielding blank or poor sequencing results.

 




MCF Home SDSU CSUPERB Contact Us Last Updated: 5/2006