Colony PCR(Ex-Taq)

Preparation

• ・5′primer
• ・3′primer
• ・2.5mMdNTP
• ・10x　standard buffer
• ・MilliQ
• ・DNA template
• ・Ex-Taq

Procedure

• 1. Number colonies.
• 2. Add 5 uL MilliQ to 0.2 mL tube.
• 3. Tap a part of colony by tip and put into numberd tube.
• 4. Put into thermal cycler at 95 degrees C for 5 minutes.
• 5. While waiting, put the following into 1.5 ml tubes. Prepare tubes one more than the sample sample number for insurance.
  • 0.1μl　100uM　5′primer
  • 0.1μl　100uM　3′primer
  • 0.8μl　2.5mMdNTP
  • 1μl　10x　standard buffer
  • 2.92μl　MilliQ
  • Voltex the tube and mix well.
  • 0.08μl　Ex-Taq (0.05ul for Takara)
  • Snap the tube and mix well
• 6. Add 5ul into new tube and set for PCR
  • 1. 95 degress for 2 minutes
  • 2. 95 degrees for 30 seconds
  • 3. 52-55 degrees for 30 seconds ( depends on the melting temeprature of DNA)
  • 4. 72.5 degrees for 1minutes ×（　）kb（length of DNA）＋ as long as you are at ease
  • 5. 95 degrees for 30 seconds
  • 6. repeat 1-5 for 29 times
  • 7. 25 degrees for forever (depends on the melting temperature of DNA)