If you ran another 35 cycles you woulg get a high molecular weight smear of concanenated amplimer of no use to anyone. Generally 10 cycles of pcr is 1000 time more product and 20 cycles is 1000000 times more product. You could design 2 internal primers and run a nested pcr of first round product and a 1/1000 dilution of first round pcr product for 20-25 cycles. This would give a smaller but clean product,

Or you could dilute your pcr reaction 1:100 and run the same primers for another 15-25 cycles and see how many cycles gives you a lot of clean product without smearing.

If you have primer dimer or non specific bands in your first pcr then gel purify the correct band and run a 1:100 dilution 1ul set of samples for 15-25 cycles ( remove a tube every 2 cycles) to get a clean product.

If you really want to run more cycles of the whole original pcr then every cycle is 2x as much product. The enzyme will still be active so just re run it with no further reagent additions for another 6 cycles fo about 60 times more product

Thanks for replying again sir.

So that means if i dilute the pcr product and then run again to get better results (depending on the cycles) or else it might result in concatenated amplimer sequences which is generally of no use?

yes too many cycles resukts in smeared amplimer bands, Your pcr has now been sitting at low temperature for a long time in the presence of active enzyme so a lot of non specific priming will have taken place so your best options are now a re amplification with more cycles from original template or gel purification of the pcr product followed by dilution and re amplification of the purified pcr product

Rakhe Tepin

As Mr. Paul mentioned in the first comment, here is an article used two times amplification.

Pls refer to the following article:

DOI: 10.1186/gb-2009-10-6-r64

The section: MicroRNA profiling.

you might be putting too much template into your initial reaction and your primers and dNTPs are getting used up early. I would serially dilute the template and see if this improves yield for the first reaction.

you can also plan a nested PCR where you then run another PCR reaction with primers inside of those used in the first reaction. I have done this in the past and the first PCR shows nothing on the gel then the nested PCR shows a good band. or at least for your second round of the same primers, set a new reaction with serially diluted first reaction and see if you get better yield/bands

one last thing, check that your taq pol is good by amplifying a known to work reaction and run on a gel to confirm. I had an incident where I was not getting amplification and the taq pol had gotten contaminated (everyone in lab dipping into it) and would not amplify.

Thanks for responding everyone!

I will try what all of you suggested.