for investigate the mutation in Helicobacter pylori 23srRNA gene, the DNA samples of the bacterium were analyzed by real time pcr taqman probe.The reaction was carried out in two steep conditions under the following conditions: the reaction was performed in volume 12 with the following details:( Master MIX: 6 λ/Primer F: 0.4 λ/Primer R: 0.4 λ/Prob: 0.2 λ/H2O: 3 λ/DMSO:1 λ/DNA:1 λ)

(Hold:95c 10 min/Cycle 1: 95c 15 s/Cycle 2: 58c20 s/Cycle3: 72c 20s / Number of cycles:40) on Rotor-Gen3000 device). Annealing temperature was setted by PCR (.two double-labeled probes used are(1-FAM-TAMRA and 2-HEX-BHQ1)(Taq copenhang –denmark)( Tm company suggested are 37c and 44 c)

Results read on device that are unacceptable and illogical signal and no fluorescent light in the fam and hex channels as shown in the attached pictures(1)

The Real time pcr product was runed on gel 8% polyacrylamide and the 87bp target gene fragment was observed in all samples.these results indicate that the reaction occurs and the product is produced but the probe is not connected to target position, because the fluorescent light and the signal are not logical due to one of the following conditions1 - either the probe is connected but the reporter or quencher has a problem or 2 - the probe is flawless but unable to connect to the target site

To determine whether the probes have fluorescents was used from each of the probes 1 λ, from the main stock of probes with a concentration of 100 μmol with 1 λ (DNase I ) in the negative control samples.(The aim was to artificially break the probe by the DNase I and remove the reporter from the quencher to emit light.)

This time Fluorescent light was observed in negative control samples containing dnase, which indicates the presence of fluorescent probe and now likely the cause of the problem is that the probe not to bind to the region.

Why don't probes attach to the sequence? What solution do you suggest?

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