CTAB DNA extraction (Pinaceae)

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Method synopsis

Modified CTAB DNA extraction protocol specifically designed to isolate microbial cells from conifer needle surfaces while excluding endophytic microorganisms from internal needle tissues.

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 — Host species and geographic location shape microbial diversity and functional potential in the conifer needle microbiome (2025), Microbiome. Implementations in other papers (listed below) may differ.

1. Conifer needle tissue collection
   Collected approximately 10 g of tissue from a twig with needles from breast height from the north side of each tree using sterile razor blades and placed into sterile bags
   Quantities: 10 g tissue per tree, 67 trees total across 6 sites, 3 conifer species (31 limber pine, 18 Douglas fir, 18 Engelmann spruce)Duration: Single collection event per treeConditions: Collected from breast height on north side of trees
   Equipment: sterile razor blades, sterile bags
2. Sample preservation and transport
   Samples placed on ice and shipped overnight to University of California, Merced. Two 1-g samples were placed in 50 mL tubes, immersed in 15 mL of PBS-S buffer, vortexed for 15 s, and sonicated for 5 min
   Quantities: Two 1-g subsamples per tree, 15 mL PBS-S buffer per tubeDuration: Overnight shipping, immediate processing upon arrivalConditions: Samples kept on ice during transport
   Equipment: 50 mL tubes, PBS-S buffer, vortex, sonicator
3. Microbial cell isolation
   Transferred to new tube and centrifuged for 15 min at 3200 g. Resulting pellet consisting of needle surface microbes was resuspended in 1.5 mL of PBS, transferred to new tube, centrifuged for 5 min at 1000 g, and frozen at −80°C until extraction

Typical Equipment

• sterile razor blades
• sterile bags
• PBS-S buffer
• vortex
• sonicator
• centrifuge
• CTAB reagents

Output Measurements

• phylloplane microbial DNA

Papers Using This Protocol (1)

Species Studied With This Protocol