EMS Mutagenesis

Ben Carter, 9/25/14

Adapted from Weigel and Glazebrook, CHS Protoc., 2006

''EMS is a mutagen. All EMS steps must take place in a fume hood using bench paper, lab coat, and gloves. All materials that come into contact with EMS, including gloves and pipette tips, must be soaked in Decontamination Solution overnight. After 24h, the Neutralization and Decontamination Solutions can be poured down the cup sink at the back of the fume hood.''

EMS is only good for one year after opening.

Mutagenesis

 * 1) Add ~10,000 seeds to 50mL of 0.1% Triton X-100 in a 50mL conical vial. Cap the vial and invert it slowly five times. Pipette off as much solution as possible while minimizing seed loss.
 * 2) Wash seeds five times with 40mL of autoclaved water by inverting slowly five times for each wash. Pipette off the water.
 * 3) Add sterile water to the 20mL mark on the side of the vial. Add 20µL of 0.1M GA3 and 60µL of EMS (final concentration = 0.3%) and seal the tubeundefinedwith parafilm. EMS is readily soluble in water, but may require vigorous shaking to get it into solution.undefinedWrap the EMS bottle with parafilm and note the date on the bottle if first use.
 * 4) Rock the vial for 15h at room temperature.undefined
 * 5) Allow the seeds toundefinedsettle to the bottom of the container and pipette away the EMS solution into the Neutralization Solution.
 * 6) Add 40mL sterile water, cap and invert slowly five times,undefinedand pipette the wash into the Neutralization Solution. Rinse in this way eight times.undefinedLet the seeds soak in the last rinse for ~1 hour to allow timeundefinedfor the EMS to diffuse out of the seeds. Even afterundefinedrinsing, significant amounts of EMS remainundefinedin the seeds. Wear gloves while handling the seeds and decontaminate pipettes after use.

Generating M2 Seed Pools

 * 1) Suspend the seedsundefinedin 1.5L of 0.15% agarose and sow the seedsundefinedin 10 flats (150 mL/flat) as done typically.
 * 2) Grow the plants to maturity according to standard lab practices.
 * 3) Collect the seed progeny (M2 seeds) when the plants have reachedundefinedmaturity. Each flat should be collected separately for a total of ten genetically distinct populations.undefined This is important for identification of independent mutant alleles.

Decontamination Solution

 * 2L of 10% sodium thiosulfate
 * 20g of sodium hydroxide

Neutralization Solution

 * 400mL of 20% sodium thiosulfate
 * 4g of sodium hydroxide