D1/D2 rRNA gene sequencing (Ranunculaceae)

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Method synopsis

Yeast isolates were cultured on chloramphenicol-supplemented YM agar and identified through PCR amplification and sequencing of the D1/D2 domains of the large subunit nuclear ribosomal RNA gene using primers NL1 and NL4.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Nectar Yeasts in the Tall Larkspur <i>Delphinium barbeyi</i> (Ranunculaceae) and Effects on Components of Pollinator Foraging Behavior (2014), PloS one. Implementations in other papers (listed below) may differ.

1. Culture yeast samples for identification
   Yeast identity was determined by removing nectar from a subset of Delphinium flowers (N = 28 flowers, each from a different plant) and streaking samples onto yeast malt (YM) agar (Catalog number: Y3022, Sigma-Aldrich, Inc., Saint Louis, MO, USA) supplemented with chloramphenicol. Including chloramphenicol inhibited bacterial growth so that focus could be on yeasts.
   Quantities: N = 28 flowers, each from a different plant, one colony type per plateDuration: Standard culturing time for yeast growthConditions: Laboratory culturing conditions with antibiotic supplementation
   Equipment: YM agar, chloramphenicol, Sigma-Aldrich Y3022
2. DNA extraction and PCR amplification
   DNA from pure cultures was extracted and amplified using a REDExtract-N-Amp Tissue polymerase chain reaction (PCR) kit (Sigma-Aldrich, Inc., Saint Louis, MO, USA). PCR was performed in a volume of 20 μl using 0.8 μl of DNA, 10 μl REDExtract-N-Amp PCR Reaction Mix, 0.15 μl of each primer, and 8.9 μl of distilled water. Amplified a portion of the D1/D2 domains of the large subunit nuclear ribosomal RNA gene using the primers NL1 and NL4.
   Quantities: 20 μl PCR volume: 0.8 μl DNA, 10 μl reaction mix, 0.15 μl each primer, 8.9 μl waterDuration: Standard PCR protocol timingConditions: Laboratory PCR conditions using thermal cycler
   Equipment: REDExtract-N-Amp Tissue PCR kit, DNAEngine Thermal Cycler, primers NL1 and NL4

Typical Equipment

• YM agar
• chloramphenicol
• REDExtract-N-Amp Tissue PCR kit
• DNAEngine Thermal Cycler

Output Measurements

• yeast species identity
• DNA sequences

Papers Using This Protocol (1)

Species Studied With This Protocol

Places Where Used