Assembly Ladders

Procedure

Procedure was written by Meagan Lizarazo

PCR Reaction

• 100 μl reaction
  • 100 μl PCR Supermix High Fidelity (Invitrogen)
  • 1.5 μl VF primer (40 μM)
  • 1.5 μl VR2 primer (40 μM)
  • 1 μl diluted template DNA (10 ng/μl)

• Cycle 35x
• initial denature 95° 5 min
• 35 cycles
  • 94° 30 sec
  • 55° 30 sec
  • 68° 4:00 min
• final extension 68° 10 min
• 4° forever

Post PCR Cleanup: Qiagen PCR Cleanup Kit

• Elimination of PCR enzymes and dNTPs is required
• Use Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification kit]
  • Combine 200μl of PCR product with 1000μl (5X) Buffer PB
  • Transfer 1st half (600μl) to QIAquick spin column
  • Spin at 8000g 1 minute, reload the 600μl flow-through, spin again, discard flow-through
  • Load 2nd half (600μl) to same QIAquick spin column
  • Spin at 8000g 1 minute, reload, spin again, discard flow-through
  • Add 750μl Buffer PE, spin 17900g 1 minute, discard flow-through
  • Spin again 17900g 3 minutes to dry
  • Transfer column to a clean 1.7 ml tube, add 30 μl TE 10:1 (pH 8.0) heated to 50°, spin at 8000g 1 minute
  • Add a further 30μl TE, spin again
  • Reload 60μl to column, spin 8000g 5 minutes

• Measure yield with Nanodrop, expect 200-400ng/μl in 55μl