Brandon:Protocols/AMPure beads purification2: Difference between revisions
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*run PCR as needed, for barcodes it is the following: | *run PCR as needed, for barcodes it is the following: | ||
72C 3min | |||
95C 30s | 95C 30s | ||
(95C 10s; 60C 30s; 72C 2min) X2 | (95C 10s; 60C 30s; 72C 2min) X2 |
Latest revision as of 01:02, 8 August 2012
AMPure beads purification protocol 2-step (for <= 5 ng)[edit]
- Purification with AMPure beads 2 step PCR, for lower input, 5 ng and under.
- 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:
72C 3min 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