I used Pfu polymerase for the PCR reaction I had set a month ago and I got a positive band. I tried repeating the experiment with the same parameters but I couldn't get any band. I have checked the polymerase's activity by using it on different samples, and it works.
Hello Aarushi Naskar
If it's not the polymerase enzyme, it could be your primers. If you thaw and freeze your primers multiple times, it tends to degrade and lose their efficiency. Or it could be an issue with your sample (if you have tried a different sample). If you checked the polymerase activity with a different sample but the same set of primers, the issue is probably with your sample. If you could provide more details, it will be easy to find out what went wrong.
Best wishes!
Hello Aarushi Naskar
The DNA sample is likely at fault if you got bands with other samples. Overtime perhaps due to poor storage parameters the samples may be contaminated or lost.
If you say the reaction worked with other samples with the exact same chemical components and and parameters, then the issue is with your DNA sample. DNA degrades over time, and the best way to extend its shelf life for as long as possible is to freeze it and by sealing it in a vacuum packed container. What you can do is to measure the DNA concentration of the sample in question as well as the other samples that are working. You may have to repeat the DNA extraction if the concentration of the sample in question is too low.
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