Wiese Lab:Sequencing Reaction Cleanup

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.