Typical reasons for this issue could be non-optimal Tm of oligos or difficult-to-amplify-regions (such as GC-rich or highly repeated sequences). I could suggest to test 2-3 more sets of new oligos by using oligo designer tools.

You could try a dmso gradient of1% in steps up to 8% with an annealing temperature of 62c in case one product is lower specificity than the other.

It is possible that you have pseudogenes or close members of the gene family and that the primers work well for both amplimers in which case you may need to redesign your primers. It is possible also that you are just picking up a heteroduplex of a snp or sequence change in your amplimer which is running slower than the expected band so looks larger on a gel. Sequencing the bands would probably indicate where the problem is coming from. A touchdown pcr might help the expected amplimer to amplify better but both bands look strong so it may not work completely. Have you run an annealing temperature gradient. The primers may work at higher than 64 so a gradient of annealing temperature up to 72c,using less primer than you are at the moment may get better results

It is unfortunate that you have quite a bit of nonspecific bands. Besides Paul Rutland 's suggestion, you can consider performing a touchdown PCR. Here, you can set a high annealing temperature (eg. 67C) and with each cycle you can program the PCR to drop the temperature by one degree (deltaT = -1C or 0.5C). If your primers have a slightly higher specificity for your sequence, then it will get amplified before the temperature gets condusive for the non specific bands. This way you can produce more amplicons of the target sequence compared to the non specific ones.

Hi Hasan Kolkas ,

A lot has been said about primer design and Tm, which are the obvious points to review and i hope the above suggestions work for you.

In case they don't:

1. Touchdown PCR protocol maybe followed wherever possible depending on the instrument. Here the initial annealing temperature is kept more than Tm and brought down gradually in subsequent cycles of the reaction.

2. 1 μg/mL of Graphene Oxide has been shown to effectively enhance the specificity of multi-round PCRs.

3. Lastly you might elute the non-specific band from gel and purify and sequence it to confirm its origin, and approach with primer redesigning.

Hoping the best,

S.

Hello,

We are also using KAPPA dna polymerase for genotyping mice tissue sometimes. Whenever we have a problem with it , we repeat the reaction with NEB Q5 DNA polmerase. It is less error prone and you dont need to optimize tm, you can use its own tm calculator.

and please always keep in mind that, although companies put some names for polymerases etc " plant spesific" ; as long as you work with DNA and RNA it doesnt matter which product you are using. So please do not buy these extra expensive so-called plant spesific polymerases etc.

I don`t know if you are using with isolated DNA or directly from lyzed tissue. But if you are using lyzed tissue directly, some contaminants changes the stability of your polymerase as well as primer binding characteristics. therefore it might give unspesific bands.

Hello, good morning, try some of the following methods and choose the method without nonspecific band: 1. In the pcr step, reduce the volume of the extracted sample. For example, if you add 2 microliters, reduce it to 1 microliter and add 1 microliter to the volume of distilled water. In the enzyme addition step, reduce the volume of the pcr sample. For example, if you add 3 microliters of the extracted sample, add 2 microliters and add one microliter to the volume of water instead. 2. Because your extract sample may be thick, add about 50 microliters of water to the extract sample and perform a pcr. 3. In the pcr step, reduce the amount of primer, for example, if it is 0.75, reduce it to 0.50. 4. Changing the time and temperature of Ben Marie can also be effective. * Perform each of the above steps and each of the resulting steps. Perform the same method for all samples. With luck

After trying all the above suggestions, I would design a new pair of primers.

Chek your primer in silico

The problem is the specificity of your primers is not good enough. You should check the specificity of your primers by NCBI.