FANDOM


YEAST TRANSFORMATION (EPISOMAL)

Use this to transform plasmids into yeast where you do not intend to recombine the DNA into the yeast genome.


0.  Before you begin: you need overnights of the yeast strains you will be transforming, use 3ml in YEPD or appropriate media, as well as prepared 10XTE, 50% PEG 3550, salmon sperm DNA, and 1M LiOAC.


0.5: Be sure that you have a 95C and a 42C heat block or water bath powered on and available.  People often turn these off at any random time throughout the day, to “save power”, so doublecheck!  YOU CANNOT WARM THE BLOCK OR BATH WITH YOUR CELLS IN IT IT MUST BE PRE-WARMED.  It's a HEAT SHOCK


1.  For each strain, pellet 1ml yeast o/n per 4 transformations, minimum 1ml.  Use 3500 rpm for 2 minutes on eppendorf.


2. Wash in 100ul 0.1M LiOAc per ml of o/n.  This means remove YEPD supernatant from yeast pellet, add 100ul 0.1M LiOAc, resuspend by vortexing, respin (3600 rpm 2 minutes), and then remove LiOAc supernatant.  Use a P1000 to remove supernatants here.


3. Resuspend pellet in 40ul 0.1M LiOAc per initial ml of o/n.


4. Combine 230ul of TRANSFORMATION MIX, 10ul of resuspended yeast, and >1ul of plasmid per transformation reaction.


5. Vortex, then incubate at room temperature for 30 minutes.


6.  Incubate at 42C in heat block or water bath for 15 minutes.  Our 42C heat block is on Juniper's bench.


7.  Spin yeast at MAX SPEED rpm for 2 minutes.  For antibiotic selection, recover in 1ml YEPD for one hour at 30C.  For auxotroph markers this can be omitted.  Then resuspend in ~ 120ul of Baxter water for plating to selective plates.

Yeast episomal transformation calculator

TRANFORMATION MIX
Number of Reactions 1 5 10 20
ul to use of:
10X TE Buffer 20 100 200 400
1M LiOAc 20 100 200 400
50% PEG 3350** 160 800 1600 3200
DMSO 25 125 250 500
Salmon Sperm DNA, freshly boiled* 5 25 50 100
total 230 1150 2300 4600
  • Boiled means means put the salmon sperm DNA in a 95C heat block before use.  The block is on Katie's bench and the SSDNA is in -20C #2 in a labeled box- take an aliquot and discard ay unused after boiling
    • PEG 3550 is Sigma P/N 3640