Are the repeats contiguous or is there some unique sequence separating the repeats. If you just want to show the existance of the repeats is it possible to design primers between the repeats so that the amplimers are 1000, 3000 for example rather than having to amplify the longest fragment. Possibly even if the repeats are contiguous a common forward primer and a reverse primer that spans the junction between the repeat sequences but not fully within the repeat How much dna are you using. the fluorescence in the wells suggests that you are using too much dna or there is some protein in the template dna

Paul Rutland Thank you very much for your answer. I need to amplify the entire sequence for the purpose of transfecting eukaryotic cells(Toxoplasma gondii). To include the homologous arms, the amplification must start before HR1 and stop after HR2. The plasmid sequence is correct, or at least the length of the target sequence is accurate, as I have confirmed this using restriction enzyme digestion and small fragment PCR of non-repeat region.

The fluorescence in the wells might be related to the sequence length, as senior members in the lab have observed the same phenomenon, especially when the sequence length reaches 10000 bps. However, it has little impact on transfection. I also tried reducing the primer amount to decrease PCR product, from 2μL to 0.1μL, but this phenomenon did not show any significant improvement, as the attachment shows(the UV was broken that day so I can't get a clearer photo). The main issue is the presence of a band with a length of about 4500 bps, which consistently appears even when I change the annealing temperature and redesign the primers.

Thank you for the clarification Zongyue Tao .In that case the only suggestions that I can make are things that you have probably tried

1 Try amplifying less template dna.

2 Assume that any pcr product of that length has significant secondary structure ( high GC or high AT regions and use 1M final concentartion of betaine and/or add dmso . I would run a dmso gradient of 1% to 8% dmso and hope for better amplification at some concentration of dmso. If these are not available then ask if any of your colleagues has a pcr enzyme which comes with a high GC buffer which will probably contain both betaine and dmso so you could try that.

3 Since you have already designed additional primers then try a nested pcr where the first round pcr uses the outermost ( longest amplimer) primers then reamplify 1ul of a1/100 dilution of that pcr amplimer with any 2 primers that are internal to the first primer set...careful with ver amplification maybe only 20 cycles of the second round pcr since every 10 cycles is 1000 times more pcr product.

Just a thought but could the 4500bp product be a secondary structure (looped formation) of a longer linear product? just running at apparently the wrong size a bit like plasmids in different structures can run at different sizes?

Also I think that you senior colleagues are probably right about the fluorescence in the wells

Paul Rutland thank you very much for your reply. I will try the method you mentioned👍

Not much to add, Paul Rutland already gave you good pieces of advice. As an additive I've had very good results with CES (Combinatorial Enhancer Solution) that is a mixture of betaine, DMSO, DTT and BSA. You can find the recipe here: http://kerrlab.org/Public/5XCES

Sometimes it's also worth it to try different polymerases, specially in complex amplifications like yours. In my hands, Platinum SuperFi and Phusion polymerases work well with long amplicons.

What size reaction volume are you using? I used to amplify larger products for site-directed mutagenesis and found that running duplicate (or triplicate) 20 ul reactions gave me far better yields than 1 50 ul reaction. From what I recall, I had success with Vent polymerase from New England Biolabs, but are lots of high fidelity large product size polymerases on the market now.

I'd recommend that you increase the extension time. The general recommendation is 1 minute per 1000 bases. Check the instructions that came with the kit to see what the manufacturer suggests.

Good luck!