Hi, could you clarify primers stock concentration? You provide it in nmol. Is it chemical quantity - n?

Some issues:

1. What's your con of your template?

2. Your answers is repetitive for a single template?

3. What's con of your pcr product?

4. Which method of staining are you using in electrophoresis?

And everything you think can help to reach a correct answer for example proper working of pcr machine, calibrated instruments, and so on.

Regards

PCR troubleshooting is a very broad subject. You should provide more details before one can suggest possible solution.

PCR is very sensitive you may get such results when your mixture or primers polluted. Try not to add DNA sample in a place you prepare primers and you PCR master, also, add DNA sample to the tubes as the last component of the PCR reaction. Sometimes the thermo-cycler it self cannot control the heat correctly, thus you may see such results.

At last, all of these are some suggestions and PCR troubleshooting consist of a very broad subjects. Please check what conditions may change during you tried to reproduce your PCR and then decide what may affected your results.

Volume of template doesn't matter. Please focus on the concentration of template instead of volume.

You can try with green PCR buffer also.

First of all, I would like to thank all of you for the response and I have provided the information that you have asked for.

Andrei S. Babenka My stock primer concentration for frwd and revrs was 49.2 and 49.4 nmol respectively, and I have measured the working primers that is given bellow: Frwd pri: 123.9 ng/ul and Revrs prii: 144.3 ng/ul. I haven't checked stock solution.

Alireza Mordadi 1-DNA template concentrations are: 72, 74, 87, 92, 115, 118 ng/ul.

2- Yes, for single template and sometimes I have used different templates as well.

3- Sometime I get good quantity and sometimes very low like 3.2, 11.6, 16.8, 22.6, 26.5, 33,6, 36.3 ng/ul of Purified PCR product.

4- I am using EthBr staining system through electrophoresis.

Behrouz Mollashahi Md. Mahmudul Hasan

Mmmm...I always use primer quantity (chemical i.e. n) - 10-12 pmol per 25 ul reaction of each primer. Could You recalculate Your cancentrations in chemical quantity? FOr me it is more comfortable. But some times I use molar concentrations such is 400-500 nM (nanomol per litter). It is almost the same for 25 ul (i mean 10-12 pmol).

Dear Numan

I'm not there but according to your explanation, your problem is related to your stuffs.

Regarding to fixed agent you are using, if I were you, first of all I check my samplers that they are calibrated or not.

After that asking for calibration of PCR.

Regards

Andrei S. Babenka I am using 10 pmol concentration of working solution for 25ul of PCR.

Alireza Mordadi Thank you for your suggestions.

In my opinion, you should revise your working protocol in detail before performing further analyses/PCR experiments.