Plant DNA extraction

Arabidopsis DNA extractions from individual plants in microcentrifuge tubes

(Alonso-Stepanova Lab protocol)

-Place 100 µL of 1mm glass beads into microcentrifuge tubes using a home-made scoop (cut off the bottom of a microfuge tube with scissors or a razor blade and glue the bottom piece to the melted tip of a glass Paster pipet)

-Harvest (room temp. ok) individual lines (20-50 3-day-old seedlings or one average-size leaf per line) into microfuge tubes pre-filled with glass beads, wiping off the forceps between individuals

-Store tissues at -20˚C or -80˚C until needed or directly proceed to the next step

-Freeze samples in liquid nitrogen by resting them on the surface of a foil cup

-Grind frozen samples for 5-6 sec in a Vivadent shaker

-Add 250 µL of CTAB buffer

-Grind for 5-6 sec in a Vivadent shaker, place samples in a rack (it’s OK to keep them at room temperature while shaking the rest of the samples).

-Incubate the entire rack of samples for 30 min at 65˚C

-Cool samples to room temp by incubating on ice for < 1 min or at RT for ~10 min

-Add 250 µL of chloroform

-Mix the samples by shaking the tubes (watch for leaks; if leaking, mix by pipeting)

-Spin at max speed (14K rpm) for ~ 10 min

  1.   Transfer 200 µL of upper phase into a tube prefilled with 250 µL of isopropanol

  2.   Mix samples by inversion 3-4x

  3.   Spin at max speed (14K rpm) for ~ 10 min

-Aspirate supernatant being careful not to touch and suck out the DNA pellet (it is better to decant most of the supernatant by inverting the open tube, then spin briefly to collect the rest on the bottom, then aspirate the rest)

-Wash pellet with 70% EtOH

-Spin at max speed (14K rpm) for ~ 10 min

-Decant extra wash and spin to collect the remaining wash solution on the bottom (a few sec is ok, but a longer time will ensure that the pellet sticks better to the bottom of the tube)

-Aspirate the remaining wash

-Air dry the pellet (~10 min)

-Resuspend DNA in 100-500 µL of deionized H2O, dependent on the amount of the starting material and the size of the final pellet (vortex, let sit at RT for >10 min, shake in the Vivadent shaker, spin to collect any insoluble material on the bottom of the tube). Use 1ul DNA per 10ul PCR rxn.

Notes and recipes:

CTAB buffer:

1.4M NaCl

20mM EDTA, pH8

100mM Tris-HCl, pH8

3% CTAB (cetyltrimethylammonium bromide, Calbiochem, #219374, 100g)

(optional) 1% beta-mercaptoethanol (add fresh before use)

Glass beads can be bought at ~$26.00/pound from  1mm beads (#11079110) are our preferred choice, but 0.5mm beads can also be used (no need to have the beads acid-washed).  For harder-to-grind tissues (such as seedlings embedded in agar), heavier 2.5mm zirconia/silica beads can be used at 3 beads/tube (#11079124zx), but test your favorite brand of microfuge tubes first to make sure that they don’t crack upon shaking.  Our lab uses microfuge tubesfrom Genesee Scientific (#24-282) that, unlike other brands, almost never leak with chloroform and withstand harsh shaking with glass beads in the Vivadent shaker.  If using heavier zirconia/silica beads, we prefer thicker screw-cap tubes from Sarstedt (#72.692), as those never break or leak.