Hello. Someone designed primers and cycler settings for me and it doesn't work. I do hot start PCR with touchdown, I detect Babesia sp. DNA in Acari. The primers are F: GYYTTGTAATTGGAATGATGG, R: CCAAAGACTTTGATTTCTCTC. In the settings there's 98°C lid temperature and the cycler has

1. 120 s on 98°C

2. 30 s on 98°C

3. 60 s on 60 with -1°C increment each cycle

4. 60 s on 72°C

2-4 go until the annealing temperature gets down to 55

5. 10 s on 98°C

6. 30 s on 55°C

7. 20 s on 72°C

5-6 repeat 45 times, then it goes to 4°C forever.

I don't know if I have the right settings or if there's anything else wrong. I try to visualize it by agarose electrophoresis and only ladder is visible. Only other thing that I can think of would be that positive control is faulty and doesn't have any DNA.

Thanks everyone for suggestions.