News: 2011 bioblitz!

bioblitz shieldFarrell Lab undergraduate Adam Clark is leading the second annual Harvard bioblitz. Join us on May 1! More

News

Prof. Farrell co-authors new paper which answers longstanding scientific question about cause of tropics' stunning biodiversity. More

Current research

News pictureCurrent research extends the evolution of insect-plant interactions to other trophic levels through a broad collaboration in the beetle Tree of Life project.

A new research dimension in the lab concerns the acoustic signals produced for mating and territory defense. More

PCR - Programs useful for PCR

 

Hot start

This is a standard program for PCR. The lower the annealing temperature, the less specific the priming—this makes it easier for mismatched primers to bind, but also makes it more likely to get nonspecific priming. Typical annealing temperatures are 42ºC to 52ºC. In the example, the annealing temperature is 47ºC. We have also found increased yield in some cases by increasing the annealing and extension step times to 2:00 and 2:30, respectively.

  1. 95ºC for 1:00
  2. 95ºC for 0:30
  3. 47ºC for 1:00
  4. 72ºC for 1:30
  5. Go to 2, 39 times
  6. 72ºC for 3:00
  7. 4ºC forever

Touchdown

Touchdown programs drop the annealing temperature lower every few reaction cycles. This decreases the odds of significant non-specific binding, as the correct binding sites will be amplified preferentially at higher temperatures.

  1. 95ºC for 1:00
  2. 95ºC for 0:30
  3. 58ºC for 1:00
  4. 72ºC for 1:00
  5. Repeat the above three steps twice
  6. 95ºC for 0:30
  7. 56ºC for 1:00
  8. 72ºC for 1:00
  9. Repeat the above three steps twice
  10. 95ºC for 0:30
  11. 54ºC for 1:00
  12. 72ºC for 1:00
  13. Repeat the above three steps twice
  14. 95ºC for 0:30
  15. 52ºC for 1:00
  16. 72ºC for 1:00
  17. Repeat the above three steps twice
  18. 95ºC for 0:30
  19. 50ºC for 1:00
  20. 72ºC for 1:00
  21. Repeat the above three steps twice
  22. 95ºC for 0:30
  23. 48ºC for 1:00
  24. 72ºC for 1:00
  25. Repeat the above three steps twice
  26. 95ºC for 0:30
  27. 46ºC for 1:00
  28. 72ºC for 1:00
  29. Repeat the above three steps twice
  30. 95ºC for 0:30
  31. 44ºC for 1:00
  32. 72ºC for 1:00
  33. Repeat the above three steps twice
  34. 95ºC for 0:30
  35. 42ºC for 1:00
  36. 72ºC for 1:00
  37. Repeat the above three steps 18 times
  38. 72ºC for 5:00
  39. 4ºC forever

Hybrid programs

Programs can have a mixture of reaction conditions. Here is one developed by Geoff Morse for COI in bruchids.

  1. 94ºC for 1:00
  2. 42ºC for 0:30
  3. 72ºC for 1:30
  4. Go to 1, 5 times
  5. 94ºC for 1:00
  6. 45ºC for 0:30
  7. 72ºC for 1:30
  8. Go to 5, 34 times
  9. 72ºC for 5:00
  10. 4ºC forever