Springer Lab: TransformationYeast2: Difference between revisions
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(New page: 1. Grow overnight in 5ml tubes 2. Dilute to OD 0.2 and grow for 4 hours 3. Harvest cultures by centrifugation at 2000g for 5 min 4. Resuspend in 1mL of sterile water 5. Wash pellet thr...) |
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Revision as of 09:24, 17 August 2010
1. Grow overnight in 5ml tubes
2. Dilute to OD 0.2 and grow for 4 hours
3. Harvest cultures by centrifugation at 2000g for 5 min
4. Resuspend in 1mL of sterile water
5. Wash pellet three times in water (spin at 16000g between washes).
6. Wash pellets three times in 0.1M LiAc
7. Divide cells into two samples and add the transformation components: - 240 uL PEG (50% w/v) - 36 uL LiAc (1 M) - 20 uL Salmon Sperm Carrier DNA (10mg/mL) - 25 uL water - 1 ug transforming DNA 8. Vortex cells for 1 min
9. Incubate cells at 30C for 30 minutes
10. Heat shock cells at 42C for 25 minutes
11. Wash cells in water
12. Incubate cells in YPD overnight at 4C
13. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days
