Springer Lab: TransformationYeast96well

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(New page: 1. Grow overnight in deep (1.5mL) 96 well plate 2. Dilute between 1:20 and 1:50 (in practice 1:40) and grow for 4 hours 3. Harvest cultures by centrifugation at 3000 rpm for 5 min 3a. ...)
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Revision as of 17:43, 14 April 2011

1. Grow overnight in deep (1.5mL) 96 well plate

2. Dilute between 1:20 and 1:50 (in practice 1:40) and grow for 4 hours

3. Harvest cultures by centrifugation at 3000 rpm for 5 min

3a. combine pellets from multiple plates to increase cell numbers

4. Resuspend in 1mL of sterile water

5. Wash pellet three times in water (spin at 3 000 rpm between washes).

6. Wash pellets three times in 0.1M LiAc

6a. After last wash, keep 35 microL 0.1 M LiAc in each well

7. Prepare transformation mix per well (see table below)

 240 uL PEG (50% w/v)
 35 uL LiAc (1 M)
 10 uL Salmon Sperm Carrier DNA (10mg/mL)
 1 ug transforming DNA (in 30-50 microL)

8.Add transformation mix to each well with multichannel pipette

9. Vortex cells for 1 min

10. Incubate cells at 30C for 30 minutes

11. Heat shock cells at 42C for 25 minutes.

12. Spin cells down at 3 000 rpm for 5 minutes. Remove supernatant with multichannel pipette.

13. Add 600 microL of YPD per well and incubate overnight at 30C

14. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days

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