Wiese Lab:Sequencing Reaction Cleanup
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(New page: ==Sequencing Reaction Clean-up== #Remove ~10 μl oil using pipet tip. Transfer (15-18 μl) sequencing reaction to striptubes.<br> #Gently shake the Agencourt CleanSEQ bottle to resuspend...)
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Revision as of 17:38, 25 February 2008
Sequencing Reaction Clean-up
- Remove ~10 μl oil using pipet tip. Transfer (15-18 μl) sequencing reaction to striptubes.
- Gently shake the Agencourt CleanSEQ bottle to resuspend any magnetic particles that may have settled.
- 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.
- Add 52 μl of 85% ethanol to the reaction plate and invert 7 times.
- Place the stiptubes onto a SPRIplate (magnet) to separate beads from solution. Incubate at least 3 minutes.
- Aspirate the cleared solution and discard. Place yellow pipet tip over end of glass Pasteur pipet.
- 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.
- Let the rxn dry for 10 minutes at room temperature.
- Add 25 μl elution buffer (water) and incubate for 5 min at room temperature
- Take your reaction over to the sequencing center in the Biotech Center. See lab manager for username and password.