High-Efficiency LiOAc Transformation of S. cerevisiae
1) Grow cells overnight to a volume of 2 mL in YPD (1-2 x 107 cells/mL)
2) Dilute to 2 x 106 cells/mL with fresh warm YPD, and grow to 1 x 107 cells/mL
3) Spin down cells @ 3000 rpm for 3 min.
4) Dump s/n, resuspend in 1mL water. Transfer to 1.5 mL microtube.
5) Spin and wash with 1mL TE/LiOAc
6) Resuspend to 2 x 109 cells/mL in TE/LiOAc
7) Heat carrier DNA (SS DNA) to melt strands and store on ice.
8) Mix together 50 m l of yeast with 1 m l plasmid DNA and 5 m l (50 m g of 10 m g/mL) of carrier DNA.
9) Add 300 m l PEG 4000 (3350)/ LiOAc/TE.
10) Vortex thoroughly
13) Incubate @ 30° C (with agitation) for 30 min.
14) Add 35 m l DMSO
15) Heat shock @ 42° C for 15 min.
16) Spin down and wash with 1mL water
17) Dump s/n and resuspend in 100 m l water
18) Plate on selective media.
TE pH7.5
10mM Tris-HCl
1mM EDTA
pH to 7.5
To make a liter, add 1.2g of Tris to 900ml of water, add 2ml of 0.5M EDTA (pH8.0), pH the solution to 7.5 and top it off to a liter.
10x TE pH7.5 (1L)
10ml 1M Tris pH7.5
2ml 0.5M EDTA
Mix and pH to 7.5 with HCl, then top off to a liter with dH20. Filter Sterilize.
TE/LiOAc
50ml 10x TE (100mM Tris and 10mM EDTA)
50ml 10x (1M) LiOAc (dissolved in dH20)
400ml dH20, sterile filter into bottles
PEG (50%)
250g PEG 3350 in 500ml water (takes some time to dissolve)
PEG/TE/LiOAc
400ml 50% PEG
50ml 10x TE
50ml 10x (1M) LiOAc
