Protocol

Dr. Marcos Egea-Cortines cortines at mpiz-koeln.mpg.de
Mon Apr 27 07:31:03 EST 1998


Frozen Competent Yeast Protocol
Protocol from the Lab book of Ramon Serrano

Cell Preparation

1	Inoculate 5-20 mL of YPD or YSD dropout with your strain of interest
and grow overnight at 30 C with vigorous shaking.

2	Inoculate a larger volume of YPD with the saturated ON culture to an
OD of 0.2 or a little bit more.  I usually calculate 5-10 mL of YPD per
sample of frozen yeast so 100 mL of will give 10-20 samples to freeze.
3	Grow for two cell divisions (About 3 hours for our strains) at 30 C
with shaking.
4	Spin down 10' at 3.5 K, and wash with half volume of sterile water at
room temperature
5	Spin down as before and wash with 1/20 volume (all refered to original
growth volumes) of 1x LiAc/TE
6	Resuspend in 1/50 of original volume of LiAc/TE. Incubate for 1 hour
at 30 C.
7	Spin down and resuspend in 12% Glycerol 1x LiAc/TE. Aliquot 100 µL in
tubes and transfer to -70. DO NOT SNAP FREEZE, it decreases dramatically
the competence.

Transformation

1	Thaw a tube slowly in the bench.  
2	Add 10 µL of DMSO, 10 µL of carrier DNA and your DNA in a volume
smaller than 10 µL.  Add 700 µL of 1x PEG/LiAC/TE.  Vortex well
3	Incubate at 30 C for 30'
4	Heat shock at 42' for 20'
5	Spin down, discard supernatant and add 0.5 mL of YPD. Incubate for 1
hour at 30 C.
6	Spin down cells discard supernatant, resuspend in 500 µL of water or
TE and plate 1/5 to 1/10.

Step 5 can be skipped by resuspending in water and plating although the
efficiency will be lower

It is convenient for regular plasmid transformation. Probably not good
enough for integrations or other experiments requiring high efficiency.


Marcos





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