Hello dear friends. I have done several PCRs using COI Variant primers (very long COI primers... 45pb Forward, 44pb Reverse), they have like 5-8 degenerated nucleotides that we made for specific species of crabs, the issue is that we got several non specific bands in the PCR. The other situation is we perform two different temperature cycles that some experts recommend us and it works (at least the amplicon we need its there... with several other bands but its there), so we don't know what to do now... This is the Thermocycler protocol:
94°C-2min
--- 13 cycles +1°C per cycle
94°C-30 sec
45°C-1.3 min
72°C-1.1 min
--- 34 cycles
94°C-30 sec
58°C-1.3 min
72°C-1.1 min
---
72°C-10 min
And PCR conditions:
H2Omq: 12.3ul
Buffer: 2.0ul
MgCl (50mM): 2.5ul
DNTPs: 0.4ul
F` 10ng/ul: 0.8ul
R' 10ng/ul: 0.8ul
Taq: 0.2ul
DNA: 1ul
I know it looks rare but it works, we can't amplify our samples with "normal" cycles config, we already made temp grad, dna grad, MgCl grad, etc. and the best results are with that configuration.
My other option is perform another PCR using PCR product like template, but I'm not sure if its a good option.
Any suggestions about this issue?? :(
Regards!
Thank you very much Dr. Mahmoudpour.
I made a Temp Grad then a MgCl++ Grad and this is the last result (MgCl++ Grad from 0.4mM/ul to 2.0mM/ul, two species, specie one up, specie two down, ladder + 5 different concentrations). I still having non-specific bands. I'll move down the annealing time to 45 seconds.
Regards
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