Spheroplast Transformation
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| - | == | + | ==Materials== |
| + | * YPD plates | ||
| + | * YPD | ||
| + | * 1 M sorbitol 182 g/l | ||
| + | * SCE | ||
| + | ** 1 M sorbitol 182 g/l | ||
| + | ** 100 mM citric acid trisodium salt dihydrate 29.4 g/l | ||
| + | ** 10 mM EDTA 20 ml of 500 mM EDTA solution /l | ||
| + | ** final pH to 5.8 with HCl, autoclave, store at RT | ||
| + | * 2 M DTT | ||
| + | * Lyticase | ||
| + | ** Sigma L4025 | ||
| + | ** final concentration 10,000 units/ml in | ||
| + | *** 20 mM phosphate pH 7.5 | ||
| + | *** 50% glycerol | ||
| + | *** store in single use aliquots at -80C | ||
| + | * STC (per 60 ml) | ||
| + | ** 0.98 M sorbitol 58.8 ml of a 1 M solution | ||
| + | ** 10 mM Tris pH 7.5 600 μl of a 1 M solution | ||
| + | ** 10 mM CaCl<sub>2</sub> 600 μl of a 1 M solution | ||
| + | ** prepare from sterile solutions just before use | ||
| + | * STC + Calf thymus DNA | ||
| + | ** Add 50 μg/ml of calf-thymus DNA (phenol chloroform purified) to STC | ||
| + | * PEG solution (for 100 ml) | ||
| + | ** 19.6% PEG 8000 w/v (98 ml of a 20% solution Sigma P2139) | ||
| + | ** 10 mM Tris pH 7.5 (1 ml of a 1 M solution) | ||
| + | ** 10 mM CaCl<sub>2</sub> (1 ml of a 1 M solution) | ||
| + | * SOS (for 30 ml) | ||
| + | ** 1 M sorbitol (15 ml of a 2 M solution) | ||
| + | ** 7 mM CaCl<sub>2</sub> (210 μl of 1 M solution) | ||
| + | ** 25% v/v YPD medium (7.5 ml) | ||
| + | ** .0025% w/v uracil (75 μl of a 1% solution | ||
| + | ** water 7.2 ml | ||
| + | ** prepare from sterile solutions just before use | ||
| + | * Sorb-URA plates (per liter) | ||
| + | ** 1 M sorbitol (182 g) | ||
| + | ** 0.17% w/v yeast nitrogen base w/o amino acids w/o ammonium sulphate (Difco 0335-15) (1.7 g) | ||
| + | ** 0.5% ammonium sulfate (5 g) | ||
| + | ** 2% w/v glucose (100 ml of a 20% solution) | ||
| + | ** 2% agar (20 g) | ||
| + | ** water to 1 liter | ||
| + | |||
| + | |||
| + | |||
| + | ==Protocol== | ||
# Pick a single colony from a YPD plate containing the correct strain into 5 ml of YPD medium and grow overnight at 30C with shaking. Do not inoculate from a plate stored at 4C. | # Pick a single colony from a YPD plate containing the correct strain into 5 ml of YPD medium and grow overnight at 30C with shaking. Do not inoculate from a plate stored at 4C. | ||
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# Resuspend in 6 ml of STC + 50 μg/ml of calf-thymus DNA and combine into a single tube | # Resuspend in 6 ml of STC + 50 μg/ml of calf-thymus DNA and combine into a single tube | ||
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Revision as of 19:34, 15 November 2006
Materials
- YPD plates
- YPD
- 1 M sorbitol 182 g/l
- SCE
- 1 M sorbitol 182 g/l
- 100 mM citric acid trisodium salt dihydrate 29.4 g/l
- 10 mM EDTA 20 ml of 500 mM EDTA solution /l
- final pH to 5.8 with HCl, autoclave, store at RT
- 2 M DTT
- Lyticase
- Sigma L4025
- final concentration 10,000 units/ml in
- 20 mM phosphate pH 7.5
- 50% glycerol
- store in single use aliquots at -80C
- STC (per 60 ml)
- 0.98 M sorbitol 58.8 ml of a 1 M solution
- 10 mM Tris pH 7.5 600 μl of a 1 M solution
- 10 mM CaCl2 600 μl of a 1 M solution
- prepare from sterile solutions just before use
- STC + Calf thymus DNA
- Add 50 μg/ml of calf-thymus DNA (phenol chloroform purified) to STC
- PEG solution (for 100 ml)
- 19.6% PEG 8000 w/v (98 ml of a 20% solution Sigma P2139)
- 10 mM Tris pH 7.5 (1 ml of a 1 M solution)
- 10 mM CaCl2 (1 ml of a 1 M solution)
- SOS (for 30 ml)
- 1 M sorbitol (15 ml of a 2 M solution)
- 7 mM CaCl2 (210 μl of 1 M solution)
- 25% v/v YPD medium (7.5 ml)
- .0025% w/v uracil (75 μl of a 1% solution
- water 7.2 ml
- prepare from sterile solutions just before use
- Sorb-URA plates (per liter)
- 1 M sorbitol (182 g)
- 0.17% w/v yeast nitrogen base w/o amino acids w/o ammonium sulphate (Difco 0335-15) (1.7 g)
- 0.5% ammonium sulfate (5 g)
- 2% w/v glucose (100 ml of a 20% solution)
- 2% agar (20 g)
- water to 1 liter
Protocol
- Pick a single colony from a YPD plate containing the correct strain into 5 ml of YPD medium and grow overnight at 30C with shaking. Do not inoculate from a plate stored at 4C.
- Add 200-800 μl of the overnight culture to 350 ml of YPD medium in a 1 liter flask and grow 14-18 hours to a final concentration of 5 x 107 cells/ml (mid log phase).
- inoculating several different amounts of overnight culture into different flasks allows one to choose the flask which is ready at the time of measurement.
- Centrifuge 2x 150 ml at 22C 1000-1200g for 5 minutes in flat bottom centrifuge tubes.
- Wash the cells in 20 ml of sterile water
- Wash a second time in 20 ml of 1 M sorbitol
- Resuspend in 20 ml of SCE and transfer to two 50 ml centrifuge tubes
- Add 100 μl of 2 M DTT and the optimal amount of lyticase
- See below on determining the amount of lyticase
- Incubate samples at 37C for exactly 15 minutes
- Centrifuge at 200-300g at 22C for 5 minutes
- Remove supernatant by careful aspiration
- The pellet will be soft and fluffy; not all cells will be pelleted
- Gently add 20 ml of 1 M sorbitol down the side of the tube and swirl to resuspend (do not vortex)
- Centrifuge and remove supernatant, as above
- Wash again with 20 ml of STC
- Resuspend in 6 ml of STC + 50 μg/ml of calf-thymus DNA and combine into a single tube
- Yeast Artificial Chromosomes, Green E.D., Hieter, P., and Spencer F. A., chapter 5 in Genome Analysis: A Laboratory Manual, Vol. 3, Cloning Systems, Birren et al. (ed.), Cold Spring Harbor Press, New York, 1999
