The amount (μg) of DNA extracted from nematodes was determined with UV–visible spectrophotometry; the measurement accuracy of the spectrophotometer (Beckman Coulter DU 640, Beckman, Brea, CA) was verified with National Institute of Standards and Technology (NIST) DNA Standard Reference Material (SRM) 2372. RNA levels were measured on a Qubit 2.0 fluorometer with the High Sensitivity RNA Assay Kit (Thermo Fisher Scientific, Waltham MA); DNA samples containing ≤10% total RNA were considered acceptable for further analyses. Protein contamination was measured with a Coomassie Protein Assay Kit (Thermo Fisher Scientific, Waltham MA). DNA fragmentation and size of extracted DNA fragments were measured with pulse-field gel electrophoresis on a 1% agarose gel using a Sage Science PippinPulse PFGE (Beverly, MA) power supply and a Galileo Biosciences gel tank (Cambridge, MA). Polymerase chain reaction (PCR) amplification performed to test DNA quality and functionality used C. elegans-33 (link) and E. coli-specific (Margaret Kline, NIST) primer sequences designed on the Integrated DNA Technologies Web site and ordered from idtDNA.com (Skokie, IL) and Eurofins Genomics (Folsom, CA). Primer sequences are provided in Table S2 .
Protocol detail
Nucleic Acid Extraction and Validation
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Agarose
Assay
C elegans
Dna fragmentation
E coli
Electrophoresis
Nematodes
Pfge
Polymerase chain reaction
Primer
Protein
Protein a
Pulse
Sensitivity
Spectrophotometry
Corresponding Organization : National Institute of Standards and Technology
Other organizations : Gazi University
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Variable analysis
independent variables
- DNA extraction method
- RNA extraction method
- Protein contamination measurement method
- DNA fragmentation and size measurement method
- PCR amplification method
- Amount of DNA extracted from nematodes
- RNA levels
- Protein contamination
- DNA fragmentation and size of extracted DNA fragments
- PCR amplification results
- Spectrophotometer (Beckman Coulter DU 640) used for DNA measurement, with verification using NIST DNA Standard Reference Material (SRM) 2372
- Qubit 2.0 fluorometer with High Sensitivity RNA Assay Kit used for RNA measurement
- Coomassie Protein Assay Kit used for protein contamination measurement
- Pulse-field gel electrophoresis on a 1% agarose gel using Sage Science PippinPulse PFGE and Galileo Biosciences gel tank used for DNA fragmentation and size measurement
- C. elegans-33 and E. coli-specific primer sequences used for PCR amplification
- Positive control: NIST DNA Standard Reference Material (SRM) 2372 used to verify the accuracy of the spectrophotometer
- Negative control: DNA samples containing ≤10% total RNA were considered acceptable for further analyses
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