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Team:University of Alberta/Selection of Primary Transformants
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| - | Provided by Dr. Enrico Scarpella | + | Provided by Dr. Enrico Scarpella<br> |
| - | + | '''Selection of primary transformants'''<br> | |
'''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.<br> | '''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.<br> | ||
| - | '''2.''' Liquid sterilize the seed | + | '''2.''' Liquid sterilize the seed<br> |
| - | '''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 <sup>100</sup> + Nys <sup> 50</sup> + The proper Antibiotic for T-DNA selection (UoA binary vector this is Hyg<sup>25</sup> | + | '''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 <sup>100</sup> + Nys <sup> 50</sup> + The proper Antibiotic for T-DNA selection (UoA binary vector this is Hyg<sup>25</sup><br> |
| - | '''4.''' Seal plates with parafilm, wrap them in aluminium foil, stratify for 3-5 days at 4 degrees C and incubat in the light | + | '''4.''' Seal plates with parafilm, wrap them in aluminium foil, stratify for 3-5 days at 4 degrees C and incubat in the light<br> |
| - | ''''Liquid sterilization for selection of transformants'''' | + | ''''Liquid sterilization for selection of transformants''''<br> |
'''1.''' Incubate seed in 70% ethanol, 1 min. remove 70% ethanol<br> | '''1.''' Incubate seed in 70% ethanol, 1 min. remove 70% ethanol<br> | ||
'''2.''' Incubate seed in sterilization solution, 20min wiht shaking<br> | '''2.''' Incubate seed in sterilization solution, 20min wiht shaking<br> | ||
| - | '''3.''' Wash 5-10x with sterile water and sow on plate | + | '''3.''' Wash 5-10x with sterile water and sow on plate<br> |
| - | 'Sterilization solution' | + | '''Sterilization solution'''<br> |
8.5ml water<br> | 8.5ml water<br> | ||
1.5ml bleach at 5.25% sodium hypochlorite (final conc. 0.8% sodium hypochlorite)<br> | 1.5ml bleach at 5.25% sodium hypochlorite (final conc. 0.8% sodium hypochlorite)<br> | ||
1 microliter Triton X-100 (final conc. 0.01%)<br> | 1 microliter Triton X-100 (final conc. 0.01%)<br> | ||
| + | |||
| + | '''0.5x MS + 1.5% sucrose'''<br> | ||
| + | 6.45g MS salts<br> | ||
| + | 1.5g MES (o.5g/l)<br> | ||
| + | 45g Sucrose<br> | ||
| + | pH to 5.7 (5.6-5.8) with 0.5 M KOH<br> | ||
| + | to 31<br> | ||
| + | Aliquote in 500-ml bottles each containing 4 g of plant agar (final conc. 0.8%) | ||
| + | Autoclave. Store at RT. Microwave to Melt | ||
Latest revision as of 16:08, 28 May 2008
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
