Difference between revisions of "Springer Lab: TransformationYeast96well"

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Line 21: Line 21:
 
   240 uL PEG (50% w/v)
 
   240 uL PEG (50% w/v)
 
   35 uL LiAc (1 M)
 
   35 uL LiAc (1 M)
   10 uL Salmon Sperm Carrier DNA (10mg/mL)
+
   10 uL Salmon Sperm Carrier DNA (5mg/mL)
 
   1 ug transforming DNA (in 30-50 microL)
 
   1 ug transforming DNA (in 30-50 microL)
  

Latest revision as of 14:49, 14 April 2011

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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