DNA Sequencing

Sequencing Services Offered:

Self Service Sequencing Protocols
GTL Performed Reactions

Any questions on sequencing may be directed to Li Paetzold or Larry Harris-Haller.

 

Sequencing Protocols

Clients are able to perform reactions to be loaded on the ABI 3100 Automated Sequencer. To help ensure good results, the GTL recommends that you follow these protocols.

Prepare DNA template for sequencing. We have determined that the quality and quantity of your template often determines the success of your reaction. The GTL recommends Qiagen purification products for use on the automated sequencer. We recommend a template concentration of approximately 300 - 500 ng/µl. Your template concentration should not be less than 100ng/µl. To determine the concentration, take an OD260 reading of your template. We also encourage you to run a small sample (approximately 1 µg) of your template next to a 1 µg standard on an agarose gel. This will help you to determine the quality and quantity of your template. For sequencing of plasmids, you should use 500-600 ng of your DNA template. For sequencing of PCR products you should use 10ng/100 bases of product.

STEP 1

Performing Automated Sequencing Reactions with Perkin Elmer ABI Big Dye Reaction Mix* (1/4 reaction)

  • In a 0.5 ml microcentrifuge tube or in a 0.2 ml tube, mix together:
    Premix 2 µL (may be purchased from GTL - contains 5X buffer, dNTP mix, ddNTPs and Amplitaq FS enzyme)
    Template (500-600 ng) in 2 µL
    Primer (6-10 pmol) in 2 µL
    Total Volume = 6 µL
  • Overlay each reaction with 6 µl mineral oil or use hot bonnet and place in the thermocycler.
  • Cycle using the following profile:
    1. 96° 2 min
    2. 96° 30 sec
    3. Ta* 15 sec
    4. 60° 4 min
    5. Go to step 2-29 times
    6. 4° hold
    *Ta is the annealing temperature of your primer.

* from Dr. Bruce Roe, University of Oklahama's Advanced Center for Genome Technology

STEP 2

Reaction cleanup

Remove the excess dye-terminators using Bio-Rad Micro Bio-Spin P-30 spin columns, which can be purchased in the GTL. This is extremely important. Failure to remove the excess terminators will result in reaction failure. Make sure that your sample is dried thoroughly.

STEP 3

Sample drop-off

  • Bring your completed reactions to the GTL (BSBW 437). The GTL will load your samples for you. Place your reactions in the orange rack on the top shelf of the -20° freezer. Make sure your tubes are labeled well (using the least amount of characters as possible) and are readable.
  • Fill out a charge-out form.
  • Sequencing plates will be loaded Monday - Friday afternoons as needed. Up to 128 client reactions can be loaded daily. Samples must be in the orange rack before 2:00 pm on plate-loading days.
  • Your data will be available the morning after the gel was run. You will be given a printout of the chromatogram and you may transfer your data onto a computer in your lab. Data may also be retrieved from our server as described in the Computing Services page.

COST: Inquire.

 

Hints for successfully using the automated sequencer

  • Dilute DNA only in water, no TE.
  • Make sure your DNA template is pure. The GTL recommends using Qiagen columns to purify your template.
  • Make sure that your template is quantitated correctly. The sequence reactions works best when you use 400-600 ng of plasmid per reaction. Too much or too little template will cause reaction failure.
  • Make sure that you have calculated the annealing temperature of your primer (s) and perform your reactions using this annealing temperature. Annealing temperature (Ta) can be calculated for most sequencing primers by using either of the following formulas:

    Ta = 4(G+C) + 2(A+T) - 2

    or

    Ta = 81.5 - 16.6(log10[Na+]) + 0.41(% G+C) - (600/N) - 2
    where N = the oligo length

*From ABI PRISM Big Dye Terminator Cycle Sequencing Core Kit with Amplitaq® DNA Polymerase, FS Protocol. P/N 4303237, Revision B., 1998. The Perkin-Elmer Corporation. Pages 15-16.

 

GTL Performed Reactions

  • For plasmid DNA - provide staff with 400-600ng purified DNA
  • For PCR product - provide staff with 10ng/100 bases of PCR product
  • Provide 6-10pmol of primer.