In order to look into this issue u 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.....

Hopefully, your template DNA has not digested away by contaminants:

1. Did you use a commercial kit for your DNA extraction? This will generally increase your DNA purity (avoid contaminants), compared to use home-made solutions for DNA isolation.

2. Store your aliquot DNA in -20oC, instead of 4oC.

Sometimes the high concentration of DNA of the target gives you bad results. In these cases a dilution 5 or 10 times is desirable.

Have you checked for primer dimers [cross contamination] in your later experiments?

Have you identified the organisms that you are using?? cause different organism have different PCR reaction and needs different primer cycles and temperature..

regars 

In order to look into this issue u 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.....

Hi Rawan

A-My suggestion is ‘checking the primers again’.1- If you diluted new stock of primers, and after that the faint bands appeared, it may you didn’t mix them properly, it is because of the oily matter of it. 2- With freeze and defreezing of primer stock, it may doesn’t work properly.

B-If none of above is right, check the taq polymerase.

C-check the DNA template.

D-check the thermocycler.

P.S. 1-I faced this problem before, and I notified that the thermocycler didn’t work properly.2-“””I can’t recognize which one is positive and also negative controls”””

Good luck

Hye from the gel view that you have shown and the details that you have provided i think if you are using the same sample (different isolate but the same species) the main reason will be your DNA template. Try checking the concentration using nanovue etc and determine the concentration because too high and low might affect your amplfiication. However if you are using different sample (different isolate and different species) even thou you targeting the same site, optimization are needed. If its a faith bands try increasing your MgCl2 but if no amplification try doing gradient PCR again

. 

many thanks for your answer , 

Yuan-Yeu Yau .. I use promega kit and I store the samples at -20 C .

Ravi Pottathil ·no primer dimer appear with my result .

Mahmoud El-Hindi · i decrease the anneling temp. twice they give bands in most samples but its look very very faint 

Hamzah Abdulrahman Salman I am working in Humans DNA samples , by the way the same condition used for another human sample and give very good result 

about the quality of DNA samples , I use the same samples with other SNP and it give me clear bands as appear in the upper line of the attached photo in comparison to the lower line which i faced the problem in . 

Hi,

Promega kit should work fine. I can find that few of the samples are showing faint bands compared with your original assay. My recommendation would be to re-synthesize the primers (fresh stock). I too faced the same anomaly with few of my primers not giving desired results with course of time. I was conducting RNAi experiments. So get new primers and conduct a gradient PCR first for optimization.

Dear Rawan A El-Madhoun, 

Seems that primers may be a limiting factor in your in PCR reaction (has '0% PRIMER DIMERS'). Hence add some more primers and try again. Also, the DNA eluting solution in your kit may have some divalent cation chelators that can also reduce  your polymerase activity. Hence, Dilute your DNA in DNase free water and do PCR. Also you can use MgCl2 as adjuvant to overcome the divalent cation chelators.

All the best...

Hi Rawan,

Did you post the picture upside down? The 'wells'  are on the bottom.

Thanks for answering ,, 

Yuan-Yeu Yau , the picture in the correct direction.

 by the way I used ready Green mix instead of preparing master mix may it affect the band clearance ?? 

Hi Rawan,

I don't think they make a whole lot of difference if you use Green mix or preparing master mix then add a 6x dye.

On Addgene website (https://www.addgene.org/plasmid-protocols/gel-electrophoresis/), there is one description:

"Note: Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has been run, just as you would if you had not added EtBr to the gel in the first place."

I have also seen same advices from other places online. Usually, bands on the lower potion of the gel is 'fainter', compared to the bands on the upper portion of the gel (which is more 'intense'). You can test this theory by running the 'problem' samples on the upper potion of the gel, and let us know.