Hello Muhammad Hamza Qureshi,

I would recommend double checking if your reagents expired or check for contamination while doing your PCR. faint backgrounds could be a sign of contaminants getting into your gel.

Hope this helps,

Nicolas Xu

Have you tried a primer temp optimization?

Hello Nicholas Xu,

I reordered new primer stock in case stock might have been contaminated and double checked the expiration dates of other reagents. I forgot to mention all other primers are working fine. Thank you so much for your input. I really appreciate it.

Best,

Hamza

Good Day Hamza

PCR can definitely be frustrating. Since you are seeing the same thing in both your sample and negative control, let's start with considering what they both have in common. This will require multiple processes.

1. I would start with making everything fresh and autoclave your water to ensure it is free of contaminants.

2. If you are still having issues start by changing one ingredient at a time ie taq, buffer, etc. this is time-consuming and will use a few reagents but it is better than getting nothing over and over.

3. Keep all samples and reagents on ice until ready to use to avoiday degradation.

Other less common possibilities

1. Have you optimized your conditions try primer concentration you might be getting primer-dimers

2. Have you changed the annealing temp to optimize for your primers, a gradient method is great for this

3. Is your template DNA degraded?

All the best

Is the DNA polymerase part of a "ready to use mix" or to you separately add the enzyme & buffer?

Repeated freeze-thaw cycles can slowly degrade enzymes. If the buffer is separate, check if it has a precipitate - that's usually the Magnesium ions "falling out of solution". If you see a precipitate, just throw it away.

Basically, get a new tube of your polymerase and see if you can get the positive control to amplify.

You can fix this!