Hello Sonalika, assuming your bands are specific, I understand you feel you have too little product, is this what you mean by "very light bands"? If this is true, you might have started your gradient already at a too high temperature. If your primers are poor in GC content (below 40%) it is not surprising that you might need very low annealing temperature. In any case, if your PCR is specific, you can try to favour the annealing increasing the primer concentration, which can go up to 1 uM in the reaction mix.

If you are working on genomic DNA I would not increase the template over 3-400 ng, in general too much genomic template tends to inhibit the Taq polymerase. With cDNA you can start with up to 5 ug of RNA for the RT and use 2-4 ul of the cDNA mix in the PCR reaction.

Increasing the cycle number will depend on what you are doing. For cloning purposes it is not suggested since you increase the chance of introducing point mutations in your product(typically 20-24 cycles are ideal), but for a yes or no answer (for example genotyping) you can go up to 35 cycles, maybe more if the reaction is specific.

Finally, you might want to design better primers!

One can Add DMSO for removing non specific bands. Other wise, reducing the concentration of templete can also solve the problem, on needs to increase the number of cycles.  Adding a Proof reading polymerase can also help. 

Please, make it clear. Are you getting extra amplified light bands along with required band or the required band is light.

for first -

You can check your primer insilico for its specifiity, if it is binding to specific site or binding to other site as well. 

if primers are perfect, then the light bands are above or below the required bands, then changing anneling temp, increasing Mgcl2, reducing number of cycles to 30 or so, can rectify this problem.

For the second, if required bands is light, then it may be due to, low conc of template or primer. or less number of cycles.

All the best

I understand your your question right,  you may need to increase the number of cycles time for annealing and extentions.  This will require increasing the in chemicals components accordingly. I. .

Hello Sonalika, assuming your bands are specific, I understand you feel you have too little product, is this what you mean by "very light bands"? If this is true, you might have started your gradient already at a too high temperature. If your primers are poor in GC content (below 40%) it is not surprising that you might need very low annealing temperature. In any case, if your PCR is specific, you can try to favour the annealing increasing the primer concentration, which can go up to 1 uM in the reaction mix.

If you are working on genomic DNA I would not increase the template over 3-400 ng, in general too much genomic template tends to inhibit the Taq polymerase. With cDNA you can start with up to 5 ug of RNA for the RT and use 2-4 ul of the cDNA mix in the PCR reaction.

Increasing the cycle number will depend on what you are doing. For cloning purposes it is not suggested since you increase the chance of introducing point mutations in your product(typically 20-24 cycles are ideal), but for a yes or no answer (for example genotyping) you can go up to 35 cycles, maybe more if the reaction is specific.

Finally, you might want to design better primers!

all of the previous comments are valid, but it would help us ( and you) tremendously if you give us as much detail as you can for us to troubleshoot, e.g. What were your cycling parameters, what's the composition of your PCR reaction? 

Good luck, and tell us what worked out for you.

You should better explain your question. Are you not getting enough PCR product or do you mean you get light shadow bands? Please specify your cycling conditions and providethe primer sequences (they might anneal to form dimers or have a high degree of secondary structure due to backfolding)

Hi,

First u should do PCR

Second check your temperature and add your DMSO...

In order to look into this issue you have to look deep into each and every component of your PCR carefully. First check your programming for each step of PCR cycle as the faint bands are due to several reasons like insufficient number of your cycles, low extension time, low annealing time, increased annealing temperature, decreased denaturing temperature, high or low denaturation time. Apart from these issues, primer concentration, template concentration, dNTPs concentration, polymerase concentration, impurity of water and primers, high GC rich templates.. so its a multi-factorial problem. And u must look into each and every direction. And that is actually the PCR optimization.....

i guess u can adjust Mg conce along with template and primers to get optimum

Is it necessary to increase the chemical composition if we increase the number of cycles??

Parasite gene, Primer concentration: 10uM

Parameters :

Annealing temperature 45-65'C

Cycles: 35

Denaturation:95'C for 5 min, 95'C for 1 min

Annealing:45-65'C for 1 min

Extension:72'C for 1 min

Last extension: 72'C for 7 min

Infinite: 4'C

its better to redesign your primers with appropriate guidelines

It is the case for only one population. I tried the same for another population. Superb bands were coming!! Any measure for population specific sequences??????

may be one reason and also check you DNA quality, is the same procedure or kit used for both the populations???