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Sanger Sequencing

In addition to sequencing technologies available at the Biotech Center on campus, full-length plasmid sequencing is available through Plasmidsaurus (see directions below).

Biotech Center Sequencing

Note: Adjust the amount of DNA that is added according to the template: plasmid = 100-200 ng for <20 kb plasmid; PCR product = 5-10 ng per 100 bp.

See the Biotech Center website for pricing and further guidelines.

Materials

  • BigDye
  • BigDye 5X Buffer
  • BigDye and buffer can be purchased from the Biotech Center; ring the doorbell in room 1259 or email dnaseq@biotech.wisc.edu
  • Gene specific primers (10 µM)
  • Nuclease-free water
  • CleanSEQ beads (Beckman Coulter #A29151)
  • 80% ethanol
  • 96-well magnet (Kathy's bench)

Protocol

BigDye Sequencing Reaction

  1. In a PCR strip tube, add the following:

    Reagent Volume
    5X buffer 3 µL
    BigDye 2 µL
    Primer 1 µL
    DNA x µL (see note above)
    Water to 20 µL
    Total 20 µL

    Note: If a problem arises during sequencing, it is very likely that two primers were inadvertently used instead of 1. A PCR takes 2 primers but a sequencing reaction only takes 1, so you will have 2X as many reactions if you plan to sequence in both directions.

  2. Run the following thermocycler program:

    Temperature Time Cycles
    96°C 3 min NA
    96°C
    52°C
    60°C    		 15 s
    15 s
    3 min
    35
    72°C 1 min NA
    4°C Store

CleanSEQ Magnetic Bead Clean-Up

  1. Shake beads to mix and aliquot in a single tube 10 µL of beads per 20 µL reaction (plus 10% overage)

  2. Add 80 µL of cold 80% ethanol

  3. Mix by pipetting

  4. Place tube(s) in magnet

  5. Wait 30 s or until the beads have congregated on the side of each tube

  6. Carefully remove and discard the supernatant without disturbing the beads

  7. Add 200 µL of 80% ethanol

  8. Repeat steps 3-6

  9. Remove the tubes from the magnet and air dry the beads for 5 min

  10. Add 50 µL water, pipette to mix

  11. Samples can be stored at 4°C until submission

Submitting Samples

  1. Ensure that the strip tube(s) are properly labeled; place the tubes in a plastic baggie or wrap in foil and label with PI name, your name, and date

  2. Take samples to the Biotech Center at 425 Henry Mall (no need for ice)

  3. Place tube(s) in the mini-fridge in room 1259

  4. Login to the computer with your NetID

  5. Fill out the submission form and print 2 copies

  6. Place one copy in the correct cubby above the freezer and place the other copy on Kathy's desk

Plasmid Sequencing (Plasmidsaurus)

Materials

  • 8-well stripcap tubes
  • parafilm
  • plasmids (>10 µL at 30 ng/µL)

Protocol

  1. Schedule a parcel pickup with FedEx and affix the parcel label to a FedEx envelope or submit the sample via the dropbox at room 318 (across from the Kimble lab) in the Hector F. DeLuca Biochemistry Building (420 Henry Mall).

  2. Submit a plasmid sequencing order through Plasmidsaurus. You will need to create an account if you do not already have one. Enter the plasmid names in the exact order they will be deposited into the 8-well stripcap tubes. Also include the FedEx tracking number on the order form where indicated.

  3. For dropbox submissions: there will be a small styrofoam box next to the ice maker (should be labeled Plasmidsaurus). Samples get picked up at 1:00 pm on Thursdays and should be labeled with your PI name, your name, and date. Time your drop off so that the samples are not sitting out too long. Include a copy of the submission form with your sample and put an additional copy on Kathy's desk.

  4. Use the Qubit to measure the concentration of each plasmid and normalize each plasmid to 30 ng/µL with nuclease-free water. Add at least 10 µL of each plasmid to a tube in a 8-well stripcap tube. Label the samples by writing the order label on the first tube and the strip number on the second tube of each strip. No additional labeling is necessary.

  5. When all plasmids for sequencing are ready and aliquoted into the strip tubes, use parafilm to seal the caps and ensure they do not open during transit. Place the strip tubes and order sheet in the FedEx envelope and prepare for pickup.

  6. Plasmidsaurus will email the results in 3-5 business days.