What do your controls look like? My guess is that either your primers are making non-specific products (this is either due to too low an annealing temp or bad primer design) or they weren't designed for the genera you are using them on. Multiplex PCR is tricky to optimize - always use a full set of controls.

Hi Samarah Alwash

It seems your PCR makes some primer dimer of some no specific targets. You can redesign new primer sets for your targets to improve your results.

Thanks

In Duplex or Multiplex PCR, non-specific bands will appear with lots of factors such as poor primer specificity and efficiency, incorrect annealing temperature, quality of the template and handling error!

Here, gel image clearly shows that this is a non-specific amplification (product 350 bp and 650 bp). Because, all the wells appeared with primer dimer except sample 2 and 3. For example, if you use two set of primers with different size of products and you get amplification for a gene. Logically, you should get primer dimer for another gene. But, there is no primer dimer in sample 2 and 3. I guess the concentration of primer (160bp) lower than the primers (100bp). Try PCR by adjusting primer concentration and see! Also check primer quality! Always include positive and Non Template Control (NTC) samples in PCR reactions. I hope it will help u to get proper amplification in your duplex PCR.

these Banda are non-specific bands and not a real PCR products. you need to check whether your primers are good enough ? or most likely to form primer dimer? check your primer sequences by. a specific program or a software. Second, try a temperature gradient on your PCR to identify the optimal annealing temperature for you primers and always use a control for your PCR. Hope it helps!

Hello Samarah Alwash ,

Here is what i feel to add to the list of suggestions you already got:

1.I feel you need to check the genotypic variance of the genes, as the 650bp band is common on both sample 1 and 4

2.Analysis of what went wrong for sample 5 to give amplification could give you some useful insights

3. As primer utilization is low (from visible primer dimers) for samples 1, 4 and 5, the 350 and 650bp bands are most likely non-specific, and for that you need to check with temp gradient, additives or new sets of primers.