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Brandon:Protocols/AMPure beads purification2
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==AMPure beads purification protocol 2-step (for <= 5 ng)== *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
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