(last update : 15-05-2009)

Sequencing protocol for BigDye 3.1 on ABI 3130XL genetic analyzer

  1. Cleaning of PCR product

  2. You need 1,25 l cleaned PCR product for each sequencing reaction. If you have to sequence several reactions of one PCR product, adjust the volumes.

    For each reaction : pipet into a reaction tube :

    Mix, spin down and incubate 15 minutes at 37C, followed by 15 minutes at 80C to inactivate the enzymes.

  3. Cycle sequencing

  4. Bring into a reaction tube the following products :

    cycle sequencing :
    29 cycles : 30 seconds 96C - 15 seconds 50C - 2 minutes 60C

  5. Precipitation

  6. (These samples can be stored at -20 C)

  7. Loading of the samples

Dilution of the BigDye mix

8 times diluted reaction mix :

Use 4 l of this for each sequencing reaction.
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