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.
Hi Kateřina Bechníková ,
Have you checked the integrity of your DNA?
Have you checked the papers for similar PCR reactions? I mean both the reaction mixture, and PCR regime.
If everything is OK above, do a primer BLAST on NCBI for your primer specifity.
Good luck!
I have a different protocol:
1. Initial denaturation: 95°C for 2 min.
2. Denaturation: 95°C for 30 sec.
3. Annealing: 55°C for 30 sec.
4. Extension: 72°C for 1 min.
5. Repeat 2, 3 and 4 36 times.
6. Final extension: 72°C for 10 min.
Annealing temperature should be adjusted for your primers (about 5 C below your primers Tm). If you have some problems like primers dimers, you can raise the temperature.
Have you tried running gel on just the extracted DNA products and its quality? For temperature profiles, if the primers used were obtained from other studies, then just minor tweaks from the reported temperature profile should do the work. Good luck!
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