Brandon:Protocols/AMPure beads purification
Jump to navigation
Jump to search
AMPure beads purification protocol (for >= 5 ng)[edit]
- Purification with AMPure beads
- 1. Add 50ul beads to 50ul reaction (or 1 volume to 1 volume), mix 10 times, R.T for 8min
- For purifications with custom transposomes, add 1.5X Nuclease free H2O to sample, and consider (sample + H2O added) as the new total volume for determining how much beads to add.
- can do 1.8 uL beads to 1 uL PCR product, will retain more DNA sample
- 1.8 to 1 uL keeps DNA above 100 bp ish usually
- 1.0 to 1 uL keeps DNA above 200 bp ish usually
- 0.8 to 1 uL keeps DNA above 300 bp ish usually
- adjust ratios accordingly
- 2. Place on magnetic stand, edges of tubes lying down if want.
- 3. After 5 minutes, remove supernatant, wash with 180ul fresh made 80% EtOH twice.
- 4. Remove from magnetic stand. Air-dry for 15min (NOW DO NOT AIRDRY 8/21/2013), pipet out EtOH after 15 minutes with gel tip, re-suspend with 20 ul Qiagen EB (or nuclease free H2O), mix 10 times so all beads are resuspended, let sit at RT for 2min, then place in magnetic stand.
- 5. After 2 minutes, transfer 20 ul liquid from DNA from beads, and concentrate samples in vacufuge if needed.
NOTES:
- it was found air drying doesn't make a different in yields, and leaving tube open while finishing wash from others is sufficient.
- When running beads on TBE gel, it is found with low elution volumes, (~10 uL) there is much (~30%) sample still retained on the beads. Thus eluting more and concentrating sample recovers more sample.