Ensure all samples are shipped in dry ice and are clearly labeled. Pacific Biosciences recommends resuspending your samples in either water or 10 mM Tris-HCl. Please see the table and list below for additional DNA quantity and quality requirements and recommendations.
Minimum Quantity Needed (per Library)
250 bp to 3 Kb insert library preparation
250 ng - 2 µg
3 - 10 Kb insert library preparation
2 µg - 5 µg
10 - 20 Kb insert library preparation
10 µg - 20 µg
Recommended Characteristics of DNA Suitable for Single-Molecule Sequencing
- Minimal DNA quality: OD 260/280 and OD 260/230 should be 1.8-2.0
- Must be double-stranded. Single-stranded DNA will not be made into a SMRTcell template in the template preparation process and can interfere with quantitation and polymerase binding.
- Has undergone a minimum of freeze-thaw cycles.
- Has not been exposed to high temps (> 65°C for more than one hour can cause a detectable decrease in sequence quality).
- Has not been exposed to pH extremes (< 6 or > 9).
- Does not contain insoluble material.
- Does not contain RNA.
- Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.
- Does not contain chelating agents (e.g., EDTA), divalent metal cations (e.g., Mg2+), denaturants (e.g., guanidinium salts, phenol), or detergents (e.g., heme, humic acid, polyphenols).