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PacBio Sample Prep FAQ

The Pacific Biosciences instruments perform sequencing in real-time using single-molecule primary (not amplified) material. At this time library inputs include DNA or cDNA.
Since the instrument uses primary material it is critical that the samples submitted be in very good condition. Here are some suggestions for submitting the best possible sample and thus ensuring the best possible outcome.
As always, please send us your questions, suggestions, and feedback - this is an evolving page. Email us at DeepSequencingCoreLabs@umassmed.edu.

DNA Samples are submitted as GENOMIC DNA, AMPLICON DNA or cDNA.

  • Genomic DNA should be high molecular weight and free of nicks.
  • Amplicon DNA should be the result of log phase amplification (don't max out those cycles and reagents!)
  • The cDNA samples should be generated without nicks or small fragments (we recommend the Evrogen Mint-2 Kit or similar style first strand synthesis)
  • Nicks can be repaired using the PreCR Repair Kit. Please do not use this if you are submitting for base modification detection.
  • Samples should be in EB or water.

The Core offers conversion to cDNA (through the MBCL) if you prefer to submit RNA. If you desire the Core to perform the DNA/RNA extraction, please contact us to discuss the project ahead of time.

How much material do I need to send? Current recommendations are:

  • Iso-Seq™ (strand-specific RNA-Seq) requires 2-5 µg Total RNA
  • A cDNA library (of 500bp-1.5kb) requires 400-800 ng of cDNA
  • A long-insert genomic library (of 1.5-30kb) requires 2-5 µg of genomic DNA
  • An amplicon library requires 300-700 ng of amplicon DNA

How much depth/how many SMRTcells will I need?

  • If you are unsure about depth, please discuss your project with the Core or with a technical specialist from Pacific Biosciences. (We can refer you to the appropriate person.)
  • Please note that the predicted number of cells needed is based on the amount of target DNA. If the sample is contaminated with DNA from other organisms, more cells will be needed to reach your target depth.

How do I avoid nicking or damaging my sample DNA?

  • Minimize Free-Thaw Cycles
  • Do not expose to pH extremes
  • Avoid vigorous vortexing or syringing
  • Do not expose to prolonged high temperatures
  • Do not expose to intercalating dyes or UV radiation

What is "good" DNA?

  • Does not contain insoluble material
  • Does not contain RNA
  • Has an OD260/280 of 1.8 to 2.0
  • Does not contain EDTA or other chelating agents
  • Does not contain divalent cations like Magnesium or Manganese or Calcium
  • Does not contain denaturants like guanidine or phenol
  • Does not contain detergents like SDS, Triton, Trizol reagent, etc.
  • Does not contain contaminating material from starting tissue like heme from blood