I tried many PCR amplification conditions but the primer did not work out. The following are the PCR conditions tried.
1. 1 cycle of 94 degree celsius for 2 minutes
40cycles of 94 degree celsius for 15 seconds, 37 degree celsius for 30 seconds and 72 degree celsius for 2 minutes
1 cycle of 10minutes at 72 degree celsius
2. 2 initial cycles of 94degree celcius for 40 seconds, 45 degree celsius for 1 minute, 72 degree celsius for 1 minute
30 cycles of 94 degree celsius for 40 seconds, 52 degree celsius for 1 minute, 72 degree celsius for 3 minutes
1 cycle of 70 degree celsius for 5 minute
3. 1 cycle of 94 degree celsius for 2 minute
40 cycles of 94 degree celsius for 15 seconds, gradient of 32-42 degree celsius for 30 seconds, 72 degree celsius for 2 minutes
1 cycle of 72 degree celsius for 10 minutes
4. 1 cycle of 94 degree celsius for 2 minutes
40 cycles of 94 degree celcius for 15 seconds, 18 degree celsius for 30 seconds and 72 degree celsius for 2 minutes
1 cycle of 10minutes at 72 degree celsius
5. 1 cycle of 94 degree celsius for 2 minutes
40 cycles of 94 degree celcius for 15 seconds, 16 degree celsius for 30 seconds and 72 degree celsius for 2 minutes
1 cycle of 10minutes at 72 degree celsius
6. 1 cycle of 94 degree celsius for 2 minutes
40 cycles of 94 degree celcius for 15 seconds, 25 degree celsius for 30 seconds and 72 degree celsius for 2 minutes
1 cycle of 10minutes at 72 degree celsius
Note: Tm of primer was 23 degree celcius
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