The first question is, should they take out of the lyophilized state with a special solution such as formamide? Based on the experience that we used to remove the TaqMan probes from the lyophilized state with distilled water, we also removed this type of probe from the lyophilized state with distilled water. Does an error occur?Second question: In the project I am working on, the probe for the internal control gene (GAPDH) is TaqMan type, which is labeled with JOE dye, and the target mutation probe for identifying single nucleotide polymorphism (SNP) is PNA-FIT type, which is labeled with thiazole orange dye. According to the articles, the excited dye identification for FIT probes is in the annealing stage, but TaqMan probes happen in the extension stage. Does this interfere with the results obtained? Because we put fluorescent light in the annealing step.Another problem I faced, so far, I have done many real-time PCR runs with different devices such as StepOne Plus, HealForce, and Rotor-Gene, but the results are different. In the StepOne device, a suitable fluorescent curve is not obtained and the detected light intensity is very low and below 0.4. In the HealForce device, sometimes I get an answer and sometimes no detection happens. In the Corbett device, the intensity of fluorescent light is very low. All items such as temperature, time settings, and the amount of ingredients in Mastermix are fixed. At the beginning of the work, we put the temperature gradient and MgCl2 and primers with SYBR-Green dye. Finally, we chose the best temperature and material amounts to continue working with the target probes.At first, we thought that maybe the FAM channel of the StepOne device could not detect the excited orange thiazole fluorescent dye well, so we also checked with other devices. But except for HealForce, where we got answers sometimes, we didn't get good answers at other times. Is there a problem with the setup or the FAM channel devices cannot detect this fluorescent dye?
The length of the target product is 137 bp, and the temperature and time defined for the devices are as follows: 4 minutes 95°C initial denaturation and then during 40 cycles of 95°C, 15 seconds, 56°C, 40 seconds, and 72°C, 30 seconds. Also, getting fluorescent light is in the annealing stage or 56°C. The samples are also genomic DNA that have concentrations of about 20 to 40 ng/µl and the ratio of 260/230 is less than 1 and the ratio of 260/280 is between 1.5 and 2.
