Hello,
I'm using the classic Folmer LCO1490 and HCO2198 primers for a venerid bivalve ADN for two months but the results are still negative, I can't see the bands.
1. I tried to change the annealing temperature : 48°C (Folmer et al.,, 1994) and 50-55°C (Kappner & Bieler, 2006)... No bands
2. I tested the Taq Polymerase and and the Reaction Buffer with the DNA and my colleague's primers, I showed the band.
3. I use the reaction Buffer x5, comprises 5mM dNTPs, 15 mM MgCL
So the PCR cocktail recipe for 20 ul was :
- Buffer : 5 ul
- Primer 1 : 2 ul
- Primer 2 : 2 ul
- Taq Poly : 0.5 ul
- DH2O : 7.5 ul
- ADN : 3 ul
Cycling program : 3min (95°C), 10s (94°C), 45s (48°C), 45s (72°C) and 10min (72°C) => 38 cycles
All this do not performs good, Did anyone can help to solve my problem please?
Thank you in advance for your help !
Technically if your buffer is 5x then you should use 4ul but 5 should work. Are you getting a small size diffuse band of primer dimer. You do not give the absolute amounts of primer and ADN so some possibilities are that there is too much primer which removes Mg and the pcr fails. Also possible is that there is too much ADN which can have pcr inhibitors which also remove Mg and again the pcr fails. To get going I would add Mg up to 2.5mM and different amounts of ADN in the assay....Try adn at .5ul,1ul,2ul and 4ul and see if any concentration works.
Change the 94c step to 30 secs to ensure complete dissociation of the ADN at each cycle. If this fails then titrate the primers in a pcr using .5ul,1ul,2ul and 4 ul with all other parameters the same and 2.5mMMg. Are these newly purchased primers or have they been stored for a long time and inherited from another researcher/project? Are they dissolved in water or TE? Primers do not last forever and may have degraded. This is a strong possibility if other primers have worked on your ADN and with your reagents
Thank you very much for your response and for your advice and suggestions.
The DNA was preserved in TE for almost a year and the primers were purchased in January 2020, the lab was closed due to the pandemic and I returned to work in September.
I will try all the proposals you have advised me and I will see.
Thank you for your Help !!
Good luck Hajar Bouzaidi . One suggestion I forgot was ,if possible, borrow a primer set from someone in the lab that should work on your ADN. Just to check that the ADN and the PCR reagents are working well. It is possible if the ADN was not frozen that it has become degraded so when you are doing your reagent tests set up 3 or 4 dna samples to identify one good ADN to use while you are optimising the reaction conditions.
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