Are the 2 bands close together in size and happen most often when the amplification is strong?

If so you may just be generating a heteroduplex band during the annealing step of the pcr of 2 very similar sequences but forming a different shape so running oddly. If it is just an annoying additional band try running a temperature gradient or a dmso gradient of 0-8% dmso and see if the unwanted band disappears

@Paul Rutland

I added a gel image to my question. The bands are not that close to each other. The second band is faint. But I'm afraid it would interfere with sequencing results.

Thank you for your suggestion. I'll try adding DMSO.

I agree that is not a heteroduplex band. Hopefully either a temperature gradient or dmso gradient will work or possibly cutting out and purifying the correct band

Double bands with matK primers are a common occurrence in many plant species, and there can be several reasons for this. One possible reason is the presence of mitochondrial DNA, which can amplify along with the matK gene. Another possible reason could be the presence of introns or alternatively spliced transcripts, which can lead to multiple bands.

To prevent double band formation, one approach is to optimize the PCR conditions, including the annealing temperature, extension time, and concentration of template DNA. It is also important to use a high-fidelity polymerase to minimize errors during amplification.

Additionally, you can try modifying the primer design to target a more conserved region within the matK gene. This can be done by aligning the matK sequences from different plant species and identifying the most conserved regions. Another option is to design primers that target a different gene altogether, such as rbcL, which is commonly used for plant DNA barcoding.

In cases where double bands are still observed, it is recommended to sequence the PCR products to confirm their identity. This can help determine whether the bands are indeed from the matK gene or from other sources.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE