The simplest way would be to add more of your PCR reaction to each lane; I would recommend running at least 15 µl given the intensity you see. Remember, PCR from gDNA is going to produce far less product than most other reactions given the low number of targets at the start.

Run 4 more cycles of pcr. Your result looks clean so more cycles should increase the yield without bringing up mis primed bands

It seems counter-intuitive, but you may want to try diluting out your gDNA. Sometimes a 1:10 or 1:100 dilution will give brighter bands due to less inhibitors in the DNA.

Another possibility would be to perform a nested PCR. Dilute your PCR product 1:10 or 1:100, then perform the same PCR again with the same set of primers. Usually gives great results!

What do you want to do with your PCR products? Sequence them? Clone them? Even low intensity bands are useful. If you are sequencing/cloning, be careful about increasing the PCR cycle number too high (more than 35) or performing nested PCR as PCR is not error proof.

Paul Rutland Thank you for your suggestion. I increased the number of cycles, now I am getting intense PCR bands compared to the gel picture that I posted with the question.

Amy Klocko I want to send the samples for sequencing. I increased the number of cycles (37 cycles) and got intense bands. I am attaching the picture of the products. I wanted to ask you that can I send these PCR products for sequencing?

Yes, those look like nice crisp single bands, they should work well for sequencing. Depending on the sequencing company you either do the cleanup and quantification yourself or pay for that sample cleanup. Nice work!

Amy Klocko Thank you for your feedback :)

I will suggest dilute the PCR product 1:100 and then use this PCR product as template and do PCR with the same conditions. it will work.

Good luck

In general for high bands intensity, you have to increase the number of cycles during PCR reaction (this will increase the yield) and inject a moderate volume in the gel. Be sure that you are following the standards regarding to gel concentrations. Furthermore I think you would better to measure the concentration by nanodrop this will determine your confidence for further sequencing reaction.

Good luck

slight increase in your dNTPs conc along with the increase in pcr cycles

Arunabha Khara It is good that you have sequenceable amounts of pcr product with the extra cycles but for the future you may have to ask yourself why 37 cycles are necessary. I think that Katie A Burnette is correct and that your dna has pcr inhibitors reducing the efficiency of the pcr so that it needs extra cycles so if this continues to happen then amplifying less template dna will probably be the answer

I always wanna make sure I have clear bands for my sequencing. Increasing the number of cycles works, checking the concentration of my dna sample in most cases i dilute it works and also lowering the anealing temperature has worked countable times for me

Edwin Wanjofu How much did you lowered the annealing temperature from your expected annealing temperature?

Arunabha Khara You can move below by one degree... If it es you double bands, make it by 0.5 degrees

Arunabha Khara following the NEB "standard" protocol and using the annealing temperature calculated for a particular primer set is a nice start but will not always get you an ideal result. Each new PCR performed in your lab needs to be optimized. Minimally, this should involve running the reaction at a variety of annealing temperatures starting 1 degree higher than the calculated temperature and then working down from there in 0.5 to 1 degree increments with identical samples. Many modern PCR machines will do this for you with the "gradient" function. If you still are not getting a single bright band going to a lower annealing temperature might help, but generally will cause more problems. Many other factors like Mg2+ concentration, dNPT or primer concentration, specialized polymerases, and certain PCR additives for difficult templates (very long, GC-rich, or repeat rich) can be adjusted and/or added to further optimize. Source DNA for the template should also be clean and pure. Excess salts, protein, organics or other contaminants in your DNA samples can be a significant problem as well. There is a science to this, but also a little bit of an art. Lastly, if a PCR is requiring more than 35 cycles for quality bands or is inconsistent, optimization is definitely a good idea. Clean, easy to read sequences at the end is worth the upfront effort on the PCR.

I would suggest three possible amendment in the pcr products and i do hope it will enhance the intensity of faint bands on gel.

1. Increase the number of amplification cycles

2. Redesign the prime

3. Increase template concentration

There may be reasons for this. First, inhibitors in the extracted DNA (high chances if DNA is extracted by manual method). Optimize the annealing temperature or increase the Mg in the reaction (necessary for binding). Finally, you may perform PCR again with this PCR product as template.

Try with different annealing temperatures

1. Use temperature Gradient to obtain a strong intensity band.

2. Check the DNA quality before Going for PCR. if your template concentration is too low, Increase volume of Template.

Increase number of cycles to 38-40.

I agree with Srinivas Chaganti.

1. gradient PCR, check the dilutions of the primers

2. Increase the time of Ta

3. Increase the cycle of amplification.

*Check your DNA samples with implen or nanodrop the quality of your samples (260/230, 260/230)

* Consider the product of PCR with the efficiency of taq for include in 2