Plant tissue genomic DNA extraction for high molecular weight DNA (Brassicaceae)

Knowledge graph centered on Plant tissue genomic DNA extraction for high molecular weight DNA (Brassicaceae) with 24 nodes and 95 connections. Top connected: Atriplex canescens, Young inversion with multiple linked QTLs under se, Arabidopsis, Boechera stricta, Boechera.

Method synopsis

Extraction of high molecular weight nuclear DNA from etiolated plant seedlings with minimal organellar contamination. Uses nuclei isolation followed by DNA purification for downstream genome sequencing.

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 — Young inversion with multiple linked QTLs under selection in a hybrid zone (2017), Nature Ecology and Evolution. Implementations in other papers (listed below) may differ.

1. Population sampling and seed collection
   Seeds of B. stricta ecotypes were collected initially surface sterilized with ethanol (95%) for 2 min followed by treatment with 15% sodium hypochlorite solution (with 1% drop of Tween-20) for 25 min on a rotary shaker at room temperature. Surface sterilized seeds were thoroughly washed multiple times with sterile water and were suspended in 0.1% agar. Seeds were kept for stratification in a cold room at 4°C for 14 under dark conditions.
   Quantities: Seeds collected from multiple populations across western North America, N = 83 genotypes totalDuration: Stratification for 14 days at 4°CConditions: Cold room at 4°C, dark conditions for stratification
   Equipment: ethanol, sodium hypochlorite solution, Tween-20, rotary shaker, sterile water, agar
2. Raising aseptic seedling cultures
   To raise aseptic seedlings of LTM and SAD12 ecotypes, surface sterilized and stratified seedlings were inoculated in 250 ml flasks containing 50 ml of sterile half-strength MS liquid medium (pH 5.7) with 0.5 g l-1 MES and 1% sucrose. About 70–80 seeds were inoculated per flask. To obtain etiolated seedlings, the flasks were covered with aluminium foil and kept on a bench top rotatory shaker at 110 rpm for 20 d at room temperature.
   Quantities: 250 ml flasks with 50 ml medium, 70-80 seeds per flask, 0.5 g l-1 MES, 1% sucroseDuration: 20 days on rotary shakerConditions: Room temperature, 110 rpm shaker, covered with aluminum foil for etiolation
   Equipment: 250 ml flasks, MS liquid medium, MES, sucrose, aluminum foil, rotary shaker

Typical Equipment

• MS liquid medium
• MEB buffer
• commercial blender
• TE buffer

Output Measurements

• high molecular weight genomic DNA

Papers Using This Protocol (1)

Species Studied With This Protocol