You have a strong primer dimer at less than 100bp so you can use a hot start enzyme and also use much less primer to get rid of this band. To get rid of the others choose an annealing temperature that works and set up a gradient of dmso so tube 1 has no DMSO.tube 2 has 1% final concentration of dmso,tube 3 has 2% dmso up to 8%dmso and it is likely that at some concentration of dmso the product will be clean and you will still have a good yield and at concentrations above this level may lead to less product. If this fails then redesign the primers but dmso often works well in cleaning up pcr reactions

Check the specificity of the primer using a primer blast

increase the annealing temperature up to 65

decrease the extension by 5 seconds.

@Govindkumar Balagannavar, will annealing temp of 65 work? As my reverse primer Tm is 65 and forward primer Tm is 59.

@Paul Rutland

Thank you for the suggestion.

But I don't have your suggested reagents in our lab and it will take another 1-2 months to get them.

Is there any other troubleshooting suggestion that you would like to add?

I have 8 primer sets. None of them are working on previous study protocol.

Tonmoy Chowdhury selection of the primer pairs Tm is not the appropriate one, the difference among the primers is too high, check the below link for the primer design.

https://www.youtube.com/watch?v=JgMJL7mEdcM

You used an annealing temperature of 61 in one of the experiments, I assume 585bp was amplified at this temperature. As a result, it will work at annealing temperatures ranging from 63 to 65 degrees Celsius. At this temperature, non-specific amplification might be reduced.

check the below article for primer designing tools.

https://www.sciencedirect.com/science/article/abs/pii/S0168165621002777

Ask around other labs. Any grade of dmso will work it does not need to be top quality. You can do a hot start without using a hot start enzyme .For a manual hot start reaction make up the pcr mix without enzyme to a volume of 23ul (example). Warm all the tubes and mix to 80c and when at temperature add 2ul of diluted enzyme to every tube of a preheated enzyme in diluted buffer mix then cap the tubes and go straight to the denaturing step of the pcr reaction....a 5 minute hold at 80c at the start of the pcr program should be enough....the important thing is not to let primer and enzyme mix at a low enough temperature to let primer dimer form. Us ehalf the amount of primer that you are currently using.

Your other failed pcrs are a worry. When 8 primer sets are not working then either your primer storage is dreadful, they are made up in water and not TE, one of your other reagents is spoiled....old ntps perhaps or your pcr machine is inaccurate at one temperature .

If your other sets are not working Then use a fresh aliquot of primer, use a borrowed NTP set,buffer and enzyme and using a dna sample that has amplified with any previous primer set run a pcr cycle with the denaturation temperature set 1c higher than you normally use and the annealing temperature 3c lower than what you expect to work. Also use Mg at 0.5% higher than your usual amount.

This will ensure that the pcr works and you will get dirty amplified bands but once you have some amplification then you know that the enzyme,ntp,primer,dna and buffers all work and then you can increase temperatures,lower the Mg in order to get a clean band but when a pcr just does not work then troubleshooting is hard because too many things can cause pcr failure.

If you get no amplification with the other primer sets on 2 or 3 good quality dna samples (OD260/280 of1.8 or above) then you may have to consider ordering new primer sets....diluting these in TE and store in small volumes frozen to avoid too much freeze thawing.

If anyone at your institute freezes cell lines they may have DMSO Tonmoy Chowdhury

@Paul Rutland

Thanks a lot. I will try.