Brandon:Protocols/AMPure beads purification2: Difference between revisions

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>Bsos
(Created page with " ==AMPure beads purification protocol== *Purification with AMPure beads 2 step PCR *1. After PCR, add 25ul beads to 25ul reaction (or 1 volume to 1 volume), mix 10 times, ...")
 
>Bsos
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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 and let dry for a few minutes.
*3. After 5 minutes, remove supernatant and let dry for a few minutes.


*2nd round of PCR
*2nd round of PCR
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  1  uL of adaptor2 (barcode)
  1  uL of adaptor2 (barcode)
  23 uL nuclease free H2O
  23 uL nuclease free H2O


*run PCR as needed, for barcodes it is the following:
*run PCR as needed, for barcodes it is the following:
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  72C 3min
  72C 3min
  15C hold
  15C hold


*second round beads purification
*second round beads purification
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**6. air-dry for 15 minutes, pipet out leftover EtOH with gel tip, re-suspend with 15ul 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.
**6. air-dry for 15 minutes, pipet out leftover EtOH with gel tip, re-suspend with 15ul 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.
**7. After 2 minutes, transfer DNA solution from beads (leave a little so don't pick up beads.
**7. After 2 minutes, transfer DNA solution from beads (leave a little so don't pick up beads.


*can do size selection after if needed
*can do size selection after if needed

Revision as of 23:21, 17 April 2012

AMPure beads purification protocol

  • Purification with AMPure beads 2 step PCR


  • 1. After PCR, add 25ul beads to 25ul 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
  • 2. Place on magnetic stand, edges of tubes lying down if want.
  • 3. After 5 minutes, remove supernatant and let dry for a few minutes.


  • 2nd round of PCR
  • add the following to above dry tube:
25 uL KAPA HF mix
1  uL adaptor1
1  uL of adaptor2 (barcode)
23 uL nuclease free H2O


  • run PCR as needed, for barcodes it is the following:
95C 30s
(95C 10s; 60C 30s; 72C 2min) X2
(95C 10s; 62C 30s; 72C 2min) X3
72C 3min
15C hold


  • second round beads purification
    • 1. add 90 ul beads to above tube and mix
    • 2. let sit at RT for 8 minutes
    • 3. place into magnetic stand
    • 4. let sit for 5 minutes and take out supernatant
    • 5. wash twice with 80% EtOH
    • 6. air-dry for 15 minutes, pipet out leftover EtOH with gel tip, re-suspend with 15ul 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.
    • 7. After 2 minutes, transfer DNA solution from beads (leave a little so don't pick up beads.


  • can do size selection after if needed