PacBio sample guidelines
Genomic DNA sample guidelines
For other sample types (amplicons, RNA/cDNA, plasmids), please contact the LGTC for advice
Please provide high molecular weight (HMW) DNA, HMW DNA is >30kb in average fragment length. Since conventional agarose gels are not suited for assessment of HMW DNA we recommend using pulsed-field gel electrophoresis (PFGE) or a Femto Pulse system (Agilent) when available. We have a FEMTO Pulse system available for HMW electrophoresis.
Some suggested kits for HMW DNA extraction:
– Nanobind DNA extraction kits (PacBio)
– Puregene Kits (Qiagen)
– Monarch HMW DNA Extraction Kits (NEB)
For quantity measurements, please use a Qubit system. A nanodrop does not provide accurate quantifications. For RNA, we recommend RIN scores of 7 and higher.
Handling and storage of HMW gDNA:
– Since you are working with HMW and wish to keep physical shearing to a minimum, please avoid vortexing (mix with wide-bore pipette tip instead), avoid pH<6 and pH>9, avoid high temperatures and minimize freeze-thaw cycles.
– Do not store DNA at 4°C for longer periods (>2 months). For long term, storage, at -20°C or -80°C is highly recommended.
– Do not use ethidium bromide for gel extraction. If DNA extraction from gel is necessary, use SYBR Safe (Thermo Fisher) instead.
– Avoid over drying of gDNA when using precipitation methods.
– DNA should be eluted in neutral, buffered solution (e.g., 10 mM Tris Acetate or Tris-HCl, pH 8) and stored in TE (10 mM Tris,pH 8, 1mM EDTA).
Purity of gDNA:
– DNA should be free of contaminants and RNA. RNAse treatment is highly recommended and most extraction kits include this step. Contamination from the organism or the extraction procedure should be avoided as much as possible as it can interfere with the sequencing of native DNA. Measuring the OD 260/280 and 260/230 ratios with a spectrophotometer can indicate if the sample contains contaminants. Clean gDNA samples typically have an OD260/280 >1.8 and an OD 260/230 of 2.0 – 2.2.
Please contact info@lgtc.nl to obtain a quote, project number and sample submission form. You can also contact us with any questions you have about your project.
Before submitting your samples you are required to fill in a sample submission form. Send it back to info@lgtc.nl
Fill in the sample submission form (SSF). Follow the instructions on the form.
Label samples clearly. Use stickers where possible.
Note:
– We don’t accept non-readable handwritten names
– Use specific sample names (if not possible, use projectcode_1, projectcode_2 etc.)
Put your samples + print-out of the sample submission form in a plastic bag. Put the LGTC project number on the bag too.
Send a digital copy of the sample submission form to info@lgtc.nl
Bring the samples to R05-02, use the checklist to see if your samples are ready for drop-off.
Fill in the drop-off form and put your samples at the correct location (4°C/-20°C) or find an LGTC employee to store the samples in case of -70°C.