Hi, I am currently trying to amplify the mitochondrial nad5 region of mosses. However I am getting highly inconsistent pcr amplification results.

I am following a published protocol, and the process was easily standardised.

But within the space of 2-3 days I started getting hazy bands & soon no bands at all, except the ladder- sometimes not even the controls.

I have checked that am not missing any of the components, all the chemicals are working fine & the gDNA are giving excellent bands when checked in 1% agarose gel.

I have already successfully amplified the chloroplast rbcl gene region of the same samples only 2 months back.  But now rbcl, its, trnl-f, nad5 - none are working.

I’ve tried diluting the samples, changing conc. of some chemicals & PVP.

Once I got results with its primers & 1/20 dilution. But the results proved non-reproducible…now I am back to square one.

Any help or suggestions would be highly appreciated.