Long Range PCR using NEB Master Mix - Hot Start Taq was working fine for me (amplicon sizes of ~10kb) but stopped working all of a sudden. Is it possible that many freeze-thaw cycles on the DNA template introduces nicks into the DNA strand that hamper the polymerase activity ? In certain cases, I am getting no amplification and in some I am getting very weak amplification. Because the integrity of the DNA should be intact over the entire length in case of long range PCR, am I right in thinking nicks in DNA will be especially detrimental if we are trying to amplify large fragments ?
The concentration of the DNA templates I used was 90-140ng/ul in a 10ul PCR reaction, DNA eluted in TE buffer after a magnetic bead method of DNA extraction.