Difference between revisions of "VR/VF2 Gradient PCR Experiment"

Revision as of 17:14, 28 August 2007

I tried gradient PCR to see if a higher annealing temperature would prevent the incorrect primer binding.

Experiment

I prepared 8 reactions for both S03582 and I13033

PCR Reaction

• 9 μ? PCR Supermix High Fidelity (Invitrogen)
• .25 μL of 40 μM VR
• .4 μL of 25 μM VF2
  • I ran out of 40 μM VF2 so I used more of a less concentrated sample to make up the difference
• 1 μL template DNA (~10 ng/μL)

• Initial denature 95°C - 5 min
• 35 cycles
  • 94°C - 30 sec
  • Gradient from 54°C to 70°C - 30 sec
  • 68°C - 36 sec
• Final extension 68° - 10 min
• 4°C forever

*The annealing temperature gradient ran from 54°C to 70°C, but due to the placement of 8-well strips samples were at the following annealing temperatures: 55.4°C, 56.7°C, 58.5°C, 60.9°C, 63.6°C, 65.8°C, 67.6°C, 68.7°C

I ran the results on a 1% Agarose gel for 30 minutes.

Results

Results of Gradient PCR

The extra bands still appeared at the different annealing temperatures.