Matt:LabNotes/Bead Purification Protocol: Difference between revisions

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>Mzcai
(Created page with "==AMPure beads purification protocol (for >= 5 ng)== (copied from Brandon's protocols) *Purification with AMPure beads *1. Add 50ul beads to 50ul reaction (or 1 volume to 1 ...")
 
>Mzcai
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**0.8 to 1 uL keeps DNA above 300 bp ish usually
**0.8 to 1 uL keeps DNA above 300 bp ish usually
**adjust ratios accordingly
**adjust ratios accordingly
**Source: http://www.keatslab.org/blog/pcrpurificationampureandsimple
*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 for better efficiency) 80% EtOH twice.
*3. After 5 minutes, remove supernatant, wash with 180ul (fresh made for better efficiency) 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, 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.
*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 DNA from beads (leave a little so don't pick up beads), 1ul left in tube for Qubit measurement

Latest revision as of 22:50, 3 July 2013

AMPure beads purification protocol (for >= 5 ng)[edit]

(copied from Brandon's protocols)

  • Purification with AMPure beads
  • 1. Add 50ul beads to 50ul reaction (or 1 volume to 1 volume), mix 10 times, R.T for 8min
    • 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
    • Source: http://www.keatslab.org/blog/pcrpurificationampureandsimple
  • 2. Place on magnetic stand, edges of tubes lying down if want.
  • 3. After 5 minutes, remove supernatant, wash with 180ul (fresh made for better efficiency) 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.
  • 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