I'm using the Q5 system and I'm PCRing a product that will have a final length of around 9500 bases. I have noticed that the product is there but very faint. The primer seems to be used up.
So my question is, can the dNTPs be rate limiting in a long reaction? That is, can all the dNTPs be used up in the first PCR cycles and maybe not be available for the later exponential rounds?
It seems you have about 1000 times the amount of dNTPs to the amount of primer (200 µM compared to 0.2 µM). So maybe...or is my basic chemistry off. Any precedence?