Pengenceran bertingkat dilakukan terhadap sampel semen. Pengenceran dilakukan dengan mencampurkan semen dengan dH2O dengan persentase 100%, 10%; 1%; 0,1% 0,01%; dan 0,001%. Setiap tingkat pengenceran diteteskan ke kertas saring dan cotton swab sebanyak 10 µl untuk dijadikan noda semen. Uji Acid Phosphatase (AP) dilakukan di setiap tingkat pengenceran. Hasil positif terakhir uji AP didapatkan pada konsentrasi sampel noda semen 0,1%. Ekstraksi DNA sampel node semen dengan variasi konsentrasi 0,001--10% dilakukan dengan metode ekstraksi chelex 20%. Hasil ekstraksi DNA sampel noda semen dikuanntifikasi dengan RT-PCR. Kuantifikasi DNA menggunakan Quantifiler Human DNA Quantification kit. Konsentrasi minimum noda semen didapatkan dengan menghitung hasil kuantifikasi DNA menggunakan pendekatan regresi linear. Konsentrasi minimum noda semen untuk menghasilkan 1 ng DNA bernilai 38%.

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The influence of semen stain concentrations on DNA quantities observed at 5 dilution rate. Semen was diluted on dH2O to obtain 100%, 10%, 1%, 0.1%, 0.01%, and 0.001% semen concentrations. 10 µl of semen solutions were then drops on top of whatman paper and cotton swab to attain semen stains sample. Acid phosphatase screening test show positive result can be observed up to 0.1% semen concentrations. DNA extraction on semen stain samples performed by chelex 20% methods. Extracted DNA were then quantified by RT-PCR. The quantification processes do by utilizing Quantifiler Human DNA Quantification kit. Minimum concentrations of semen stains needed to attain optimum DNA quantities analyzed by calculating the linear fit regression of semen concentrations vs. DNA quantities curve. The result shows minimum semen stain concentration to produce 1 ng/µl DNA was 38%.