Microbotryum silenes-dioicae
. Year of collect: 2001 Country of collect: Switzerland Locality (name of city): Gampel Ploidy: N Sexual type: A2 Host plant: Silene dioica
Title
MvSd-72-A2-G1
Strain
72
Molecule
genomic_DNA
Extract_protocol
proto-extraction-fungi_EN_v1.pdf Document author : Helene Badouin Date : 2013/03/25 This documents describes the protocol of DNA extraction which was used in Giraud s lab for DNA extraction of Microbotryum violaceum. The protocol was adapted by Alodie Snirc from a protocol of the Worden lab (cf file CTAB DNA Extract_GenomeSeq quality.pdf), adapted by Evelyne Derelle at Observatoire Oceanologique, Banyuls sur Mer from Winnepenninckx B. et al., 1993, TIG: 9 (12), 407 (Technical Tips).  * Cell harvesting : Cells are harveted from Petri dishes cultures and transferred in 2 ml tubes.  We harvest half a plate to a complete plate depending of the amount of material. Tubes are either processed at once for DNA extraction or stored at -20°C up to a few weeks.  * DNA extraction : -PL1 (Macherey-Nagel) pre-heated at 60°C -Chloroform-isopropanol 24:1 -Sterile micro-beads -Liquid nitrogen -Freeze tubes in liquid nitrogen for a few seconds. -Add 800 µl PL1 and homogenize with a pipet if necessary. -Crush twice 30s at 30 Hz. -Add 15 µl of RNAase. Gently mix by inverting the tubes. -Incubate tubes at 60°C at 150 rpm agitation for 3-4 hours. -Transfer the liquid into new tubes of 2 ml and discard the old tubes with the beads. -Add 800 µl Chloroforme-isoamylalcool in the fume hood. Mix for 2 minutes by inverting the tubes. -Spin 15 min at 15300 rpm, 4°C -Carefully transfer the aqueous phase (above the white interface layer) to a clean 2 ml tube (then discard the rest). Be conservative to avoid contamination by chloroform. -Add 10µl RNAase in mix by inverting the tubes. -Incubate at 37°C with agitation (150 rpm) for 30 min. -Add 600 µl isopropanol at -20°C and gently mix by inverting the tubes several times -Store tubes overnight at -20°C -Spin 15 minutes at 15300 rpm. -Discard supernatant -Wash with 500 µl of 70% ethanol kept at -20°C. Mix the tubes so that the pellet is detached from the tube. -Spin 15 minutes at 15300 rpm. -Discard supernatant -Dry the pellet at room temperature for 30 min. -To eliminate traces of ethanol, incubate open tubes at 37°C for 30-40 min. -Resuspend the pellet in 20-40 µl of TE (elution buffer) depending on the size of the pellet. -Incubate at 37°C for 1 hour. Mix and make a short spin a few times during this step.  *Quality Control and dosing : -Nanodrop Make blank with the elution buffer. Measure 230/260 and 280/260 ratio.  Dosing is not precise because it measure both DNA and RNA. -1% agarose gel Dilute 1 µl of DNA in 99 µl of TE. Load 10 µl DNA + 4 µl cyanol blue. 5 µl Smart Ladder. Migration 20 min at 135 V. Check that DNA is not degraded. -PCR Sl14 to check that the DNA belongs to Microbotryum :  * Fluorometer dosing : DNA were dosed with a fluorometer Qubit 2.0 from Invitrogen. -Dilute 1 µl of Qubit reagent in 199 of buffer to make the Qubit working solution. -Make standart solutions by diluting 10 µl of standarts (2 tubes) in 190 µl of working solution -Dilution 1µl of DNA in 199 µl of working solution. -Vortex all essay tubes for 2-3 seconds. -Incubate at room temperature for 2 min. -Read tubes in fluorometer.