Hi, I am building a custom PCR device, I want to design some PCR templates to test the thermocycler but i do not know which is the right concentration for the DNA.
I am grateful if you can tell me how to choose the right concentration because I have no knowledge about the biochemistry.
The concentration of DNA template depends on the source. Normally used concentration are 100-250 ng for mammalian genomic DNA and 20 ng for linearized plasmid DNA (circular plasmid DNA is slightly less efficiently amplified) per 50µl reaction.
Use high quality, purified DNA templates whenever possible. Please refer to specific product information for amplification from unpurified DNA (e.g., colony PCR or direct PCR);
For low complexity templates (e.g., plasmid, virus, BAC DNA), use 1 pg–10 ng of DNA per 50 μl reaction;
For higher complexity templates (e.g., genomic DNA), use 1 ng–100 ng of DNA per 50 μl reaction;
Higher DNA concentrations tend to decrease amplicon specificity, particularly when a high number of cycles are run
You can see the range is quite broad. Usually for small genomes you would use lower concentrations, since you expect more copies of your target sequence per ng.
Test some published PCR primers for mouse housekeeping genes like GAPDH or Beta actin. For template, use genomic DNA from mouse tails from any research lab. Genomic DNA prep of 80-100 ng/µl concentration is fine and you can use 400-500 ng of total template DNA for a 25 µl reaction to test the thermal cycler. You may use our published article below for as a good reference and use same primers to just test the thermal cycler if this is what you want to do. Remember these are mouse primer sequences.
I think you have got your answer.
1. For plasmids: 10 to 20 ng of template DNA.
2. Yeast 200 ng to 1ug.
3. For others 50 to 200 ng template DNA.
4. Always use purified DNA.
5. For initial use housekeeping gene primers like actin etc.
In theory 50-100 ng gDNA is used for gDNA and for plasmid DNA 10-20ng.But in practical case you may need less or more DNA.So,you should prepare different concentrations of template and find out in which concentration you find best product means prominent band without any traces of primer dimers.
Minimum visual detection limit of NA is 0.5 to 5.0 ng by ethidium bromide staining. Copy number is dependant on size. Therefore theoretically, 1 copy ( 0.5 ng) can be multiplied by PCR. You can use different concentration as measured by UV , of NA primarily to set MDL by your experiment. Your purification method should be robust.
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