I am having a problem with my standard curves.
I am running a duplex reaction looking for 2 C. diff genes. I am using the ABI7500 Fast and Qiagen QuantiFast PCR kit. One primer/probe set is FAM labeled and the other is VIC. Both have a final concentration of 500nM primer/250nM probe. I optimized both primer/probe sets and then combined them with no change in Cts. I have been running samples for a few months and the standard curve samples were working fine.
Then in the last month the FAM labeled ones started behaving odd, from 3-15 cycles the lines would jump around below the baseline causing me to have to raise the threshold. Then my highest std started coming up late, usually after the next dilution. This then throws off the whole line since the highest concentration of DNA has a higher Ct than the next dilution or two. I thought the FAM primer/probe set was going bad so had a new set made, same problem. Also, now the second set, VIC-labeled, are doing the same thing when they were not before. The DNA concentration according to the nanodrop is around 500 ng/uL and I'm adding 5uL in each well so the highest conc is 2526.5 ng/uL. I am doing 1:5 dilutions for the curve which gives a range from 2526.5-0.0065ng/uL. I have made up quite a few new std curves, changed the water used, run the reactions alone and always get the same issue. I have extracted the DNA from a spore stock using a basic heat/NaOH procedure that has worked very well and gives results with an absorbance around 1.6 which seems good for gram Positives.
Any advice???? I'm not sure what else to try since this was working and is now not and seems to be worse each time I try something. Thanks for reading.