I don't know about you but I see non-specific bands everywhere. Even in the sample #34 (I believe you meant 34). The difference between #34 and the other 6 is that number 34 has a darker 1090 band (target amplicon). From my experience with PCR I know that when the target quantity is smaller, the non-specific amplification increases. This could be the reason why you have stronger specific bands in the rest of samples.

But, if you just need to do gel extraction of your target band, you need not care. Just excise your band and throw away everything non-specific.

Yes, as it has been said you could try to change the concentration of your template and you could also try to decrease the concentration of primers. But anyway you can extract the band and look for it.

Smear looks like degradation of DNA. Re-amplification will be not successful.

Thank you for your answers everyone,

Normally I'm not worried about non-specific bands because - just as you say - I'm extracting the bands. However, these intense non-specific bands are reducing the amount of target product and are also very close to the target band. I've already had problems with low concentrations from the purified gel slices, as well as failed sequencing due to noise, so I'm concerned about contamination from the non-specific bands.

I will try using more template material and perhaps running on a higher % gel to get more separation from the non-specific bands.

you can treat this problem by cut your target band from the gel and purify this band bu gel extraction kit then use the purified product as your template in a new PCR reaction and run on the gel and see

To me this doesn't look like degradation or non-specific priming, as there is a ladder with regular intervals, not a smear. It is very similar to bands we've observed when amplifying with a primer that anneals in a repeat in the target sequence. Is it possible that your target contains a repeat?

Cornelia H de Moor , That's a good point that the bands are on regular intervals. I noticed that initially but didn't know what to make of it. My target here is the mtDNA cytochrome b gene, so there shouldn't be any repeats. I checked my primers, and they should be specific to their intended priming locations.

I had considered that something wacky is going on with the extension process, like self-priming or some 'slippage' of the enzyme, but I don't know if that's possible or how to investigate. It's strange because this protocol has worked on about 70 samples before I ran into this problem.

I've tried a few more amplifications with more template material, and got some improvement on some samples (fewer of the non-specific bands, and farther away from the target band), but it's not consistent.

Could be your primers are self-annealing and repeatedly re-priming themselves to form the ladder effect. This would happen if you have multiple complementary sites on the primer(s). Its unlikely but not impossible. Try the reaction without template to see if the primers are the cause.

Hi, Simon,

Check your primers annealing temperature and primers concentration, in most of the commercial pcr master mix needs less concentration in primer. So, dilute your pcr primers 10 times. Image showing smearing of pcr product, it mostly happed for those cases.

Good luck!