DNA Input Recommendations
For best results, follow the input recommendations. Use 500 ng input gDNA. Quantify the input gDNA and assess the gDNA quality before beginning library preparation.
Quantify Input DNA
Use the following recommendations to quantify input DNA:
- Successful library preparation depends on accurate quantification of input DNA. To verify, run samples in duplicates.
- Use fluorometric-based methods for quantification, such as Qubit or PicoGreen.
- DNA quantification methods that rely on intercalating fluorescent dyes measure only double-stranded DNA and are less subject to the presence of excess nucleic acids.
- Do not use spectrophotometric-based methods, such as NanoDrop, which measure the presence of nucleotides and can result in an inaccurate measurement of gDNA.
- Quantification methods depend on accurate pipetting methods. Do not use pipettes at the extremes of volume specifications. Make sure that pipettes are calibrated.
Assess DNA Quality
Absorbance measurements at 260 nm are commonly used to assess DNA quality:
- The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity. Values of 1.8–2.0 indicate relatively pure DNA.
- The presence of RNA or small nucleic acid fragments, such as nucleotides, can compromise both absorbance measurements.
- Make sure that samples are free of contaminants.
Use 1 or more of the following recommended positive control samples. These positive control samples can be used for methylation status control.
- HCC1187 normal (BL) (ATCC, catalog # CRL2323-D)
- NA12878 (Coriell Institute, catalog # NA12878)
- HCC1187 breast cancer tumor (ATCC, catalog # CRL2322)
- HeLA (Biochain, catalog # D1255811)
- Jurkat (Biochain, catalog # D1255815)