RPB1-F1843 5′-ATTTYGAYGGTGAYGARATGAAC-3′ and
RPB1-R3096 5′-GRACRGTDCCRTCATAYTTRACC-3′
Length amplicons 900-1000 bp
I have used these primers for identification of Talaromyces sp. with PCR conditions as follows:
Initial denaturation 94°C - 3 min
[Denaturation 94°C - 1 min, Annealing Gradient 50 - 65°C - 30s, Extension 72°C - 1 min] x 34 times.
Final extension 72°C - 5 min
Result: No bands, nothing else
So, can somebody who used to run these primers share me some experience for that, or let me know another specific primer for Talaromyces sp.
Thank you in advance.
The way oligos are made each dimorphic base is made as 2 distinct oligos.
As you have 10 redundant positions in your 2 oligos this represents 2exp10 individual oligos in your primer mix ( about 1000 oligo sequences) . Your dna template probably only has 1 or 2 of these possible sequences if it is a pure sample so you might want to try increasing the amount of primer to ensure enough of the correct primer anneals to get an efficient pcr. Possibly also run a different pcr to ensure that your dna quality is good enough to amplify generally ( does not contain inhibitors)
Thank you very much for spending your time.
Probably, I will try for increasing the concentration of the primers.
I hope that we'll keep in touch in the future.
I wish you the health, happiness and success.
Dear Minh
I had also faced this issue with number of primers and for different crops. So I’m totally agreed with Mr Paul. Moreover, run your pcr cycle st annealing temperatyre, just 2-3 degrees below the average of provided melting temperature of your primers.
Make sure your primers are purified with HPLC!
If you know the sequence of template then you could shift RPB1 upstream so it would end with 5′-?????????ATTTYGAYGGTG
and use
RPB1-R3096 5′-GRACRGTDCCRTCATAYTTRACC-3′
alternatively you may want to try the following PCR profile with 10 times primers concentration :
Initial denaturation 94°C - 3 min
[Denaturation 94°C - 1 min, Annealing Gradient 50 - 55°C for 60s, Extension 72°C - 2 min] x 5 times + [Denaturation 94°C - 30s, Annealing Gradient 50 - 55°C for 60s, Extension 72°C - 1 min] x 35 times
Final extension 72°C - 5 min
additionally, if PCR failed again - add up to 3% of DMSO (cell culture grade) to every PCR mix and repeat
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