Wiese Lab:Sequencing Reaction Cleanup

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Sequencing Reaction Clean-up

 * 1) Remove ~10 μl oil using pipet tip. Transfer (15-18 μl) sequencing reaction to striptubes.
 * 2) Gently shake the Agencourt CleanSEQ bottle to resuspend any magnetic particles that may have settled.
 * 3) Add 10 μl of CleanSEQ to the striptubes.  This step should be performed off the SPRIplate (magnet).  The same amount of beads is used regardless of the sequencing reaction volume.
 * 4) Add 52 μl of 85% ethanol to the reaction plate and invert 7 times.
 * 5) Place the stiptubes onto a SPRIplate (magnet) to separate beads from solution. Incubate at least 3 minutes.
 * 6) Aspirate the cleared solution and discard. Place yellow pipet tip over end of glass Pasteur pipet.
 * 7) Add 100 μl of 85% EtOH and incubate at room temperature for at least 30 sec. Aspirate EtOH and discard.  For sequencing rxns using 4 μl or more of BigDye, a second wash may be necessary.  A second wash may improve result quality by removing unincorporated fluorophores.
 * 8) Let the rxn dry for 10 minutes at room temperature.
 * 9) Add 25 μl elution buffer (water) and incubate for 5 min at room temperature
 * 10) Take your reaction over to the sequencing center in the Biotech Center. See lab manager for username and password.