Mucuna pruriens
cultivar
pruriens
age
seeds
dev_stage
seeds
tissue
seeds
biomaterial_provider
Julia Reimer, RWTH Aachen, Germany
disease
none
growth_protocol
Seeds were watered for 24 h to remove the outer shell, before extraction of high-molecular DNA
treatment
To extract high molecular weight DNA (hmwDNA) from M. pruriens seeds a modified protocol from Peterson et al. was used. Briefly, about 17 g seeds were soaked over night in water to remove the seed coat. Following, the seeds were ground to fine powder in liquid nitrogen using mortar and pistle. To enrich for nuclei, the powder was dissolved in 300 ml extraction buffer (2-methyl-2,4-pentanediol (1 M), PIPES (10 mM), MgCl2 (10 mM), PVP-10 (4 % w/v), sodium metabisulfite (10 mM), 2-mercaptoethanol (25 mM), sodium diethyldithiocarbamate (0.5 % w/v), L-lysine (200 mM), and ethylene glycol tetraacetic acid (EGTA (6 mM), pH 6.0). The homogenate was squeezed through 2 layers of nylon mesh (20 and 70 mikro m. The filtered homogenate was filtrated once again through nylon mesh as before. Triton x-100 was added to the beaker to a final concentration of 0.5 %, and further incubated for 30 min while gently stirring at 4 degreeC. Following, the homogenate was centrifuged at 800 x g for 20 min at 4 degreeC and the pellet resuspended using a soft brush soaked in 50 ml of MPDB (2-methyl-2,4-pentanediol (0.5 M), PIPES-KOH (10 mM), MgCl2 (10 mM), Triton X-100 (0.5 % v/v), sodium metabisulfite (10 mM), 2-mercaptoethanol (5 mM), L-lysine (200 mM), and EGTA (6 mM), pH 7.0). The pellet was gently resuspended and washed at least three times with 10 ml of MPDB (centrifugation 600 x g, 20 min, 4 degreeC), until it became clear white. After the last wash, the pellet was resuspended in less than 1 ml MPDB. To lyse the nuclei, sodium dodecyl sulfate was added to a final concentration of 2 %(w/v) and mixed by gentle inversion. The lysate was incubated at 60 degreeC for 10 min, and afterwards cooled to room temperature. Sodium perchlorate was added to a final concentration of 1 M. Following, the lysate was spun at maximum speed for 20 min and the supernatant transferred to a new tube using a wide tip to prevent DNA shearing. To perform DNA extraction, an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) was added to the nuclear lysate. This was incubated for 30 min while wrolling and then centrifuged at 3000 x g for 10 min. The aqueous phase was transferred into a new tube and second PCI extraction performed as before followed by an extraction using only chloroform. DNA was precipitated by adding sodium acetate (0.3 M final concentration) and two volumes of ethanol at -20 degreeC, pelleted and washed twice with 70 % ethanol. Last, the precipitated DNA was re-dissolved in distilled water.
country
Nigeria: Jos
BioSampleModel
Plant