I am encountering a technical issue with PCR results. I used RAPD primers (OPA 3, 7, 10; OPC-2) and ISSR primers (809, 810, 840, 841) with a 2x ready-to-load master mix (GCC Biotech), primers (G-Bioscience), and DNA extracted from plant leaves using the CTAB method. Previously, I obtained successful amplification using the same components and conditions. However, now I am not getting any bands—only the DNA ladder is visible on the gel.
I have ruled out common issues:
- Master Mix: I am using a newly procured batch.
- Primers: I switched to a new stock, but the issue persists.
- DNA Quality: The extracted DNA shows prominent bands on a 0.8% agarose gel.
- PCR Machine: My labmate successfully amplified bacterial DNA using the same machine.
Given these factors, where could the problem lie, and what steps can I take to troubleshoot and resolve this issue?
Try another set of primers (positive control), and another template DNA (ideally the one that worked previously). If the old DNA is not available, purify a new one.
Chances are, that it is the template that might be contaminated - some kits for purification of DNA (e.g. from Qiagen) include a washing step with a buffer that contains EtOH. During the final elution traces of the buffer might be left in the sample. So, when EtOH gets into the eluate it strongly inhibits downstream reactions such as restriction analysis or PCR provided you use e.g. 1 ul of the DNA per 10-20 ul reaction (personal experience). To remedy this/exclude this possibility, precipitate your DNA and dry it well before dissolving in water/buffer.
Additionally to the above answer try diluting your dna 1:2, 1:4, 1:8 and 1:16. Many dna purification methods do not completely remove pcr inhibitors and often amplifying less dna (and so less inhibitor) can improve pcr amplification
The issue is likely specific to the DNA extractions. Add in a positive control so you can be sure that everything else is working.
I'd also recommend what Paul Rutland suggests and dilute out your DNA. Plant leaves can have a lot of PCR inhibitors (e.g. phenolics). I'd try 1:10 and 1:100. Test out just a few samples (2-3), positive control, and negative control.
At least you don't have contamination!
Good luck!
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