Springer Lab: TransformationYeast96well
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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 (5mg/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
' | 1 well | 12 | 24 | 36 | 48 | 60 | 72 | 80 | 84 | 96 |
PEG 50% | 240 | 2880 | 5760 | 8640 | 11520 | 14400 | 17280 | 19200 | 20160 | 23040 |
LiAc (1M) | 35 | 420 | 840 | 1260 | 1680 | 2100 | 2520 | 2800 | 2940 | 3360 |
Salmon Sperm DNA (5 mg/mL) | 10 | 120 | 240 | 360 | 480 | 600 | 720 | 800 | 840 | 960 |
transforming DNA | 10 | 120 | 240 | 360 | 480 | 600 | 720 | 800 | 840 | 960 |
Total in microL | 295 | 3540 | 7080 | 10620 | 14160 | 17700 | 21240 | 23600 | 24780 | 28320 |
Total in mL | 0.295 | 3.54 | 7.08 | 10.62 | 14.16 | 17.7 | 21.24 | 23.6 | 24.78 | 28.32 |
For the actual master mix use 10% more reagents | 1 well | 12 | 24 | 36 | 48 | 60 | 72 | 84 | 84 | 96 |
PEG 50% | 264 | 3168 | 6336 | 9504 | 12672 | 15840 | 19008 | 21120 | 22176 | 25344 |
LiAc (1M) | 38.5 | 462 | 924 | 1386 | 1848 | 2310 | 2772 | 3080 | 3234 | 3696 |
Salmon Sperm DNA (5 mg/mL) | 11 | 132 | 264 | 396 | 528 | 660 | 792 | 880 | 924 | 1056 |
transforming DNA | 11 | 132 | 264 | 396 | 528 | 660 | 792 | 880 | 924 | 1056 |
Total in microL | 324.5 | 3894 | 7788 | 11682 | 15576 | 19470 | 23364 | 25960 | 27258 | 31152 |
Total in mL | 0.3245 | 3.894 | 7.788 | 11.682 | 15.576 | 19.47 | 23.364 | 25.96 | 27.258 | 31.152 |