You will be deluged with suggestions!

Mine is to provide more information! For example, what size fragment are you expecting PCR to generate? [reducing the distance between primers should help, as might nested PCR]

agree with @Geoff Margison

regards

Following

i agree with Geoff that the amplimer size is critical but even if only a few molecules of the right size are present then nested pcr will give about a billion billion fold amplification with 2 rounds of 30 cycles so large amounts of dna are not needed but pcr contamination will be a real problem unless you are very careful

I agree with Geoff too.

Did you use Qubit HS for measuring the concentration of DNA? Nanodrop is not a good method when you try to measure low concentrations of DNA.

If you used Qubit and your measure is precise, maybe you could try to use no more than 15ng because in highly degraded samples, this damaged DNA could inhibit the amplification (more is less in this case)

Ana is right, and what was the 260/280 ration if you did use a nanodrop? It would be good to see the absorption profile, and would also be helpful to know what your extraction method was.

What was your extraction method? Maybe you can come up with an optimized method taking steps from a variety of protocols.

I also recommend to do a touchdown PCR to see which temperature and number of cycles fits your DNA.

Hi everyone, thank you for your suggestion so far.

My goal now is collect as much as possible DNA from these samples, esspecially human DNA fragment.

@Geoff My primers now is 3 different sets, 2 fragment of 167 bp and 314 bp specific to human mtDNA and other is 187 bp for interspecies. And maybe I will try random hexamer later.

@Crista I'm using PowerMax DNA Extraction kit (Qiagen), and one commercial kit from local company. And I have tried some optimization for the PCR as gradient PCR (48oC - 60oC), increase number of cycles (30 - 45 cycles), reduce volume of primer and also increase DNA template.

DNA 260/280 ratio is from 1.4 ~ 1.8, but 260/230 ratio is around 0.

If you want to get maximum yield of DNA from your samples you should try some other extraction method. Chloroform phenol(Organic method) can provide you maximum quantity of DNA .

No method that I am aware of will extract human DNA in preference to any other species'!

It may be worth checking the size of your DNA on an agarose gel, although the amounts that you have may require a highly sensitive detection method (or if you use LMP agarose, you could recover most of it): that might tell you if the primers you are using are suitable.

You might also try serially diluting your DNA ,then PCR: my experience is that less input DNA can give huge increases in PCR products.

In case improving DNA extraction is not enough, I may suggest preamplification byusing Genomiphi or other protocols involvin Phi29 or other whole genome aplification procedure...