Brandon:Protocols/AMPure beads purification: Difference between revisions
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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 | *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 | *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.