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PCR

Here are a couple of PCR protocols for non-degenerate and degenerate PCR which have served me well. The 'TD48' ("Touch Down ending at 48") protocol starts with a high, restrictive annealing temperature (58 C) which then drops 1 degree C over the next 9 cycles, followed by 29 cycles at 48 C to accomplish the majority of the amplification. The 'TU58' ("Touch Up ending at 58") protocol has been a lifesaver for troubleshooting degenerate pcr, turning streaky lanes into nice crisp bands. There are three sets of annealing temperatures, each of which gets progressively more restrictive. The first set of four cycles anneals at 35 C, the second four at 45 C and the last set of 30 at 58 C. The temperature of the last set can easily be tweaked to optimize the reaction for the primers. Lastly, the extension times and temperatures should also be adjusted depending on the Polymerase used and the length of the amplification (general rule about 1 min/kb, although this seems to be conservative). Let me know how these work!

Non-Degenerate PCR

TD48

1. 94 C for 3:00
2. 94 C for 0:30
3. 58 C for 0:30 (decrease by 1.0 C every cycle)
4. 72.0 for 0:45
5. Cycle to step 2 for 9 more times
6. 94 C for 0:30
7. 48 C for 0:30
8. 72 C for 0:45
9. cycle to step 6 for 29 more times
10. 72 C for 10:00
11. 4 C forever

 

Degenerate PCR

TU58

1. 94 C for 3:00
2. 94 C for 0:45
3. 35 C for 0:45
4. 72.0 for 2:00
5. Cycle to step 2 for 3 more times
6. 94 C for 0:45
7. 45 C for 0:45
8. 72 C for 2:00
9. Cycle to step 6 for 3 more times
10. 94 C for 0:45
11. 58 C for 0:45
12. 72 C for 2:00
13. Cycle to step 10 for 30 more times
14. 72 C for 10:00
15. 4 C forever