Electroporation Protocol for Nostoc Punctiforme

Electroporation Protocol 1. Inocualte 2-5 days prior for a disperse exponential AA/4 ammonia grown culture. Grow in in a shaking incubator (26°C, 250RPM, 500ml culture in 1 l flask). 2. Prepare Nostoc. a. Morning of EP, take a Chla reading of culture and concentrate. Concentrate a volume of culture enough for a total Chla of [ # of EP's X volume of EP (usually 0.4ml) X 100 µg Chla/ml] down to a volume of 3-5 ml in growth medium. SonicateNostoc culture gently (10 bursts, 50% duty at 1.5 in 3-5ml). Place into 50 ml AA/4-ammonia mops for >4 hours recovery in New Brunswick or under bench. b. Just prior to EP, sterily wash cells 3X with 20 ml room temperature ddwater, vortexing between washes. After third wash, resuspend to a volume that gives a concentration of 50-100 µg/ml Chla. Cool on ice immediately before use. 3. Prepare DNA. Run minipreped DNA through a mini column prep protocol (Quiagen) omitting the incubation times. Elute with water. Use 4-20µl/EP. 4. Electroporation. Aliquot DNA to sterile microfuge tube and set on ice. Chill EP cuvettes on ice. Add 400µl concentrated, washed cells to microfuge tube, mix by pipetting up and down, and wait 30-60 seconds to allow DNA adsorption to cells. Transfer to cold 0.2cm cuvette, cover and electroporate. EP parameters which work: 600 Ω 1.6 kEV 25µF Expected time constants (MSec): Volumes which work: 400µl with 20µl DNA ~12.5-13.2 120µl with 4µl DNA ~11.5-12.5 5. Electroporation recovery. IMMEDIATELY following electroporation dilute cells in cuvette with AA/4-ammonia mops containing 20mM MgCl2 and remove to a 50 ml flask of the same media. Incubate overnignt in dim light with gentle shaking. 6. Selection. Concentrate overnight recovered culture and resuspend in ~1ml Vortex vigorously to break up clumps. Plate 100µl of this undiluted culture, as well as 1:5, and 1:10 dilutions onto selective media. Place remaining cells under liquid selection for backup. Incubate under low light until colonies are visible at ~11 days. Selection which work for Nostoc punctiforme: PSCR119/pSUN119 based plasmids 10 µg/ml Nm PSCR202/pSUN202 based plasmids 5 µg/ml Ap