If you isolated the DNA from pork meat that you obtained commercially, it is very likely that the DNA is degraded.  If your PCR product size is large (or even comparable) to the average size of the fragmented DNA in the pork DNA prep, the efficiency of amplification will be strongly affected. [At high template concentrations, you could get interactions from two overlapping fragmented molecules that result in a "PCR template repair" and a normal PCR product.  But at lower template concentrations, such interactions become geometrically more rare--and this could explain the dilution effect.] Just run the DNA on a gel with size standards and compare it to the DNA in your earlier prep--if that is possible.  You may need to obtain pork tissue from a fresher source.

Hi !

Your gDNA is getting degraded or somehow your water inhibited the PCR.

Don't dilute DNA, RNA or primers in water. For DNA you could use TE0.1x, or better some special buffer containing carrier RNA or DNA. For a quick solution just use the elution/resuspention buffer from the kit.

Inhibitors in your water, or the quantity of gDNA is too  low eluted from extration kit that you are using. For better isolation of gDNA, in some reactions you use carrier DNA and internal control without dilution and in some reactions carrier DNA and internal control with dilution. Then you will interpret your results. 

could be  degraded DNA

could be expired SybrGreen master mix, since you say you have beeing performing this protocol for a year, it could be totally expired. 

could be proteins contamination, since you have changed your extraction kit and you have not standarized the new conditions for pure DNA yet. 

since its the same g DNA of the same species, primers and annealing temp are not probably the problem. 

Go back to the extraction kit that gave good results, attach to it, sometimes things go well and we have to stick to them. 

Could be poor qaulity DNA, or presence of some PCR inhibitors

You can try PCR enhancers. See the attachment for some PCR enhancers you may interested in.

Hi Lee Lee, provided your reagents are not expired as suggested above, there are a few things you could check out.

1. Your sample's concentration could be off. Use a spectrophotometer to check your sample's concentration. Be sure to use the same diluent to blank the machine first. If you don't have a machine that can measure using very small amounts of sample, and your sample is expensive then maybe you can skip this.

2. If you've the same water to dilute the samples to get picture "AP" then there probably isn't anything to do with the water. Try running the experiment by diluting the samples with the same water and running the old and new samples in parallel in the same run on the same machine.

3. Check how your original porcine gDNA was prepared. Did they have any enzymatic digestions at all? Was the gDNA prepared in the same way as in your new sample?

4. If you are using the same primers, run the products from the old and new sample on the gel. Are you getting a product of the same size? Would be good to run this on the products from (2).

If you are still getting the same results, post the gel pictures as well as the new curves (super-imposed and separate) and we'll see what we an do next.

I am in agreement with those above that you need to use a carrier - e.g., yeast tRNA etc.  Apparently, your new extraction method makes your gDNA more prone to sticking to the plastic of your tubes.  And, the more dilute your samples become, the higher the percentage of overall sample that sticks to the plastic - leaving less and less gDNA available to the qPCR.  Given that your first (most concentrated) point is about 11 or 12 Cq in both your old and new assays, inhibitors don't really seem to be your issue.  Degradation could be an issue if you are not adding a nuclease-inhibitor somewhere (but DNase inhibitors are hard to find - whereas RNAse inhibitors are very easy to find). Does pork meat contain high amounts of DNase enzyme(s)? Or, perhaps carry-over myosin, myoglobin, polysaccharides, calcium, etc., may all collude to change the net charge of your porcine meat gDNA sample in such a way that more of the negatively- charged phosphate backbone on the gDNA is exposed, making it more and more susceptible to binding positively-charged plastic surfaces.  Adding a carrier, I believe, will help you stop this problem.  Another reason why inhibition doesn't seem to be the concern here is because the more and more you serially dilute the porcine meat-derived gDNA sample, the less and less inhibitors would be affecting your assay...  Therefore, I think the carrier addition is the right idea to pursue.  Let us know what you find~!

If you isolated the DNA from pork meat that you obtained commercially, it is very likely that the DNA is degraded.  If your PCR product size is large (or even comparable) to the average size of the fragmented DNA in the pork DNA prep, the efficiency of amplification will be strongly affected. [At high template concentrations, you could get interactions from two overlapping fragmented molecules that result in a "PCR template repair" and a normal PCR product.  But at lower template concentrations, such interactions become geometrically more rare--and this could explain the dilution effect.] Just run the DNA on a gel with size standards and compare it to the DNA in your earlier prep--if that is possible.  You may need to obtain pork tissue from a fresher source.