Hi G.J. Nouairia,

First of all the conc of your DNA is well enough for PCR reaction. Even if we use 20-30 ng/ul also, we can get good amplification. You need to optimize your PCR condition. Try following suggestions:

(1). Using inappropriate PCR cycles can lead to insufficient amplification. Optimize your PCR cycles. Try to use between 30-35 cycles.

(2). If the extension time is too short, there will be insufficient time for complete replication of the target. Generally, use an extension time of 1 min/kb.

(3). If the annealing time is too short, primers do not have enough time to bind to the template. Use an annealing time of at least 30 sec.

(4). If the annealing temperature is too high, primers are unable to bind to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. If the primer Tm minus 5°C is close to the extension temperature (72°C), consider running a two-step PCR protocol. The annealing temperature should not exceed the extension temperature.

(5). If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low. Use a denaturation temperature of 95°C.

(6). If the denaturation time is too long, DNA might be degraded. For the initial denaturation, use 3 min at 95°C; for denaturation during cycling, use 30 sec at 95°C.

(7). If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. For the initial denaturation, use 3 min to activate the polymerase; to denature the template during cycling, use 30 sec.

(8). If the dNTP concentration is too high, Mg2+ depletion occurs. Each dNTP should be present at 200 μM in the final reaction.

(9). GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. DMSO or another secondary structure destabilizer can be added (do not exceed 10%).

(10). Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.

(11). Using an excessive concentration of primers can increase the chance of primers binding nonspecifically to undesired sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.

(12). If the polymerase concentration is too low, not all PCR products will be fully replicated. The optimal enzyme concentration depends on the length and difficulty of the template.

(13). Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mM in the final reaction.

Try these suggestions. You have not mentioned about your gene size. You need to optimze your PCR according to your gene size.

Hope any of the points works for you.

Cheers.

Dear Ghada

Just take the amplified mt DNA amplified in First PCR as a template and do  PCR again. You will get more product. Even you can perform nested PCR

Hi G.J. Nouairia,

First of all the conc of your DNA is well enough for PCR reaction. Even if we use 20-30 ng/ul also, we can get good amplification. You need to optimize your PCR condition. Try following suggestions:

(1). Using inappropriate PCR cycles can lead to insufficient amplification. Optimize your PCR cycles. Try to use between 30-35 cycles.

(2). If the extension time is too short, there will be insufficient time for complete replication of the target. Generally, use an extension time of 1 min/kb.

(3). If the annealing time is too short, primers do not have enough time to bind to the template. Use an annealing time of at least 30 sec.

(4). If the annealing temperature is too high, primers are unable to bind to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. If the primer Tm minus 5°C is close to the extension temperature (72°C), consider running a two-step PCR protocol. The annealing temperature should not exceed the extension temperature.

(5). If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low. Use a denaturation temperature of 95°C.

(6). If the denaturation time is too long, DNA might be degraded. For the initial denaturation, use 3 min at 95°C; for denaturation during cycling, use 30 sec at 95°C.

(7). If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. For the initial denaturation, use 3 min to activate the polymerase; to denature the template during cycling, use 30 sec.

(8). If the dNTP concentration is too high, Mg2+ depletion occurs. Each dNTP should be present at 200 μM in the final reaction.

(9). GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. DMSO or another secondary structure destabilizer can be added (do not exceed 10%).

(10). Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.

(11). Using an excessive concentration of primers can increase the chance of primers binding nonspecifically to undesired sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.

(12). If the polymerase concentration is too low, not all PCR products will be fully replicated. The optimal enzyme concentration depends on the length and difficulty of the template.

(13). Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mM in the final reaction.

Try these suggestions. You have not mentioned about your gene size. You need to optimze your PCR according to your gene size.

Hope any of the points works for you.

Cheers.

Often, changing another polymerase does better than all other optimizations. By an unknown reason, each polymerase has some template preference. Good dNTP is also important.   

What is the size of the amplicon and the GC content?

You likely have too much DNA not too little. Sometimes DNA can carry over some inhibitors. mtDNA is 200-300 copy per cell, so you have plenty template in 5 ng or less. 

Some good suggestions were made already, so good luck.

Thank you for everybody especially Mr Gaurav Chhetri , this was very helpful. I am going to try these suggestions and then post back the solution that worked best for me. 

Dear Laurent Vanhille: the size is 1247bp and GC content is 46.7%. 

Gaurav Chhetri absolutely rite...

and may be your DNA is degraded you should confident that your DNA purify... 

@Ghada Jebah Nouairia....What were your controls?

Well Gaurav Chhetri ,

@Ghada Jebah Nouairia

if all the conditions are right then you can take, and use the amplified DNA as a template and amplify it. You can can good amplificaton i.e nested PCR

I'd love to have that much DNA! I frequently amplify from template which has undetectable levels of nucleic acid. Have you looked at your sample on a nanodrop? You may have contaminants which are inhibiting your PCR- in which case, diluting your template may help. I know it sounds wrong, but diluting the sample will dilute the contaminants and you'll still have enough DNA. If I suspect I have PCR inhibitors in my sample, I dilute it 1:1, 1:10 and 1:100. Otherwise, optimise your PCR reaction as Guarav has suggested. If I'm really struggling then I'll try different kinds of Taq, or just loads and loads of taq (Not recommended, but a desperate last measure).

Thank you again for everybody. I changed the Taq polymerase today and the PCR worked perfectly. 

I was about to suggest changing the polymerase but I can see you already tried it and got your PCR to work. In the future, I would suggest checking all you reagents for quality first before changing too many conditions of the PCR. Sometimes, it's as simple as mistakenly using the old polymerase or buffer.

@ Ghada,

What would be your thoughts about the possible reasons that the previous DNA polymerase did not work, and this new one worked? What are the possible factors contributing to the success of the most recent PCR?

The previous Taq worked with some DNA templates (purified by Phenol Chloroform) while not with others (extraction by salting out and diluted twice as much as the others). The new Taq uses new buffer and MgSO4 (instead of MgCl2). So I think some inhibitors are present in the DNA samples that didn't amplified and the new conditions where more favorable for the PCR reaction to take place.

Have you better explanation based to your knowledge and experience?

PCR could be dramatically enhanced by Au nanoparticles. With the addition of 0.7 nM of 13 nm Au nanoparticles into the PCR reagent, the PCR efficiency was increased. Especially when maintaining the same or higher amplification yields, the reaction time could be shortened, and the heating/cooling rates could be increased. The excellent heat transfer property of the nanoparticles should be the major factor in improving the PCR efficiency. Different PCR systems, DNA polymerases, DNA sizes and complex samples were compared in this study. Our results demonstrated that Au nanoparticles increase the sensitivity of PCR detection 5- to 10-fold in a slower PCR system (i.e. conventional PCR) and at least 104-fold in a quicker PCR system (i.e. real-time PCR)

for more details you can read this paper