The Pacific Biosciences RS Instrument performs sequencing in real-time using single-molecule primary (not amplified) material. At this time we use DNA or cDNA though RNA is planned as a future upgrade at some point by PacBio. 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. Send to 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.
If you drop off samples in one of our freezers in the school or LRB, please email us so we can come get them quickly. Regular sample drop off is at the MBCL lab in 2 Biotech, suite 207.
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