Team:University of Alberta/Selection of Primary Transformants


Provided by Dr. Enrico Scarpella
Selection of primary transformants
1. Aliquote ca. 100 mg of seed of the dipped plants in 3 microtubes (ca. 33 mg seed per tube, i.e. ca. 80 microliters seed per tube.
2. Liquid sterilize the seed
3. Transfer speed in each tube to 3-4.5 ml of sterile, hand warm 0.1%, mix by inversion and pour the entire contents of the tube on a 100-mm plate with ca. 25ml or 1/2 MS + 1.5% sucrose + Cb 100 + Nys 50 + The proper Antibiotic for T-DNA selection (UoA binary vector this is Hyg25
4. Seal plates with parafilm, wrap them in aluminium foil, stratify for 3-5 days at 4 degrees C and incubat in the light

'Liquid sterilization for selection of transformants'
1. Incubate seed in 70% ethanol, 1 min. remove 70% ethanol
2. Incubate seed in sterilization solution, 20min wiht shaking
3. Wash 5-10x with sterile water and sow on plate

Sterilization solution
8.5ml water
1.5ml bleach at 5.25% sodium hypochlorite (final conc. 0.8% sodium hypochlorite)
1 microliter Triton X-100 (final conc. 0.01%)

0.5x MS + 1.5% sucrose
6.45g MS salts
1.5g MES (o.5g/l)
45g Sucrose
pH to 5.7 (5.6-5.8) with 0.5 M KOH
to 31
Aliquote in 500-ml bottles each containing 4 g of plant agar (final conc. 0.8%) Autoclave. Store at RT. Microwave to Melt