Brandon:Protocols/AMPure beads purification: Difference between revisions

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==AMPure beads purification protocol==
==AMPure beads purification protocol (for >= 5 ng)==




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*1. Add 50ul beads to 50ul reaction (or 1 volume to 1 volume), mix 10 times, R.T for 8min
*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
**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
**1.8 to 1 uL keeps DNA above 100 bp ish usually
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*2. Place on magnetic stand, edges of tubes lying down if want.
*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.
*3. After 5 minutes, remove supernatant, wash with 180ul fresh made 80% EtOH twice.
*4. Remove from magnetic stand. Air-dry for 15min, pipet out EtOH after 15 minutes with gel tip, re-suspend with 25ul 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.
*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 DNA from beads (leave a little so don't pick up beads), 1ul left in tube for Qubit measurement
*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.

Latest revision as of 21:00, 21 August 2013

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.