Yeast Transformation Protocol

From 2012.igem.org

1- Prepare a 2 ml preculture in the selection medium of the strain to be transformed.

2- Inoculate 20 ml of YPD for each transformation, in order to get an OD600 = 1 next day.

inoculum vol.= (final OD)/(preculture OD)×(final vol.)/2^n

Where n is the number of divisions (generation time: 1´5 h for S. cerevisiae).

From now, it is not necessary to work below sterility conditions.

Once reached the OD600 = 1:

3- Centrifuge at 3000 rpm 5 min.

4- Wash with sterile water.

5- Resuspend in 30 ml of LISORB.

6- Shake at ambient temperature for 30 min.

7- Centrifuge at 3000 rpm 5 min and resuspend in 1 ml of LISORB. Transfer to an eppendorf tube.

8- Centrifuge at 3000 rpm 5 min.

9- Resuspend in 100 μl of LISORB for each transformation. Transfer 100 μL aliquots in different tubes for each transformation.

10- Add 7 μl of salmon sperm DNA + 1 μl of transforming DNA.

11- Incubate 10 min at ambient temperature.

12- Add 260 μl of 40%PEG/LiAc/TE. Mix well.

13- Incubate 1 h at 30°C.

14- Add 43 μl of DMSO and give a thermal shock of 5 minutes at 42°C.

15- Centrifuge at 3000 rpm 5 min.

16- Wash with 1 ml of sterile water.

17- Centrifuge at 3000 rpm 5 min.

18- Resuspend in 0´5 ml of water and plaque:

-50 μl

-Rest (centrifuge and decant leaving 50-100 μl).