CDNA clean up
From Ucsbgalaxy
Revision as of 08:56, 12 September 2012 by 128.111.227.84 (talk)
ds cDNA clean-up for Illumina sequencing (Agencourt AMPure XP)
- Vortex beads to ensure paramagnetic beads are fully suspended
- Transfer the samples to 1.5 mL tubes if necessary
- Add the appropriate volume of AMPure XP beads: multiply PCR volume by 1.8 (1.8 µL beads for 1.0 µL PCR product).
- Mix by pipetting 10 times (color should be homogeneous). Let the mixed samples incubate at room temperature for 10 minutes. This step binds PCR products to the paramagnetic beads.
- Transfer reactions to 0.2 mL tubes.
- Place on the magnet plate for 2-5 minutes to separate beads from the solution. Check to make sure solution is clear before
- Aspirate the clear solution without disturbing the beads
- Dispense 200 µL of 70% EtOH to each tube and incubate at RT for 30 seconds. Aspirate out the ethanol and discard. Repeat for a total of two washes. Do not disturb the magnetic beads.
- Incubate at RT for 5 minutes to dry the beads.
- Off the magnetic plate, add 40 µL elution buffer and mix by pipetting 10 times. Elution is quite rapid and it is not necessary for the beads to go back into solution for it to occur.
- Return tubes to the magnet plate and incubate at RT for 2-5 minutes.
- Transfer the eluent to clean 1.5 mL tubes.