I have been stucked with the problem i am going to mention hereunder.
I performed the first round PCR for PCV2. Positive and negative controls were included. Except for the positive control, all samples and negative control were negative by the 1st round PCR. Using this pcr product as the template, nested PCR was performed. Utmost care was taken to avoid any contamination by using one pipettes tips per sample although same pipettes were used for the pcr and nested pcr. My lab doesnt have separate room for PCR and nested PCR. By nested PCR all samples and negative control were positive but the positive control turned out to be negative. The result of negative control by nested PCR seem to indicate that there could have been some contamination post pcr. But what could have caused the positive control that was positive by pcr to be negative by nested PCR?