Home

About

Advanced Search

Browse by Discipline

Scientific Societies

E-print Alerts

Add E-prints

E-print Network
FAQHELPSITE MAPCONTACT US


  Advanced Search  

 
Yeast Transformation 1. Grow yeast to mid-log in YPDa (YPD + 1X adenine). Dilute fresh YPD overnight culture 1/100
 

Summary: Yeast Transformation
1. Grow yeast to mid-log in YPDa (YPD + 1X adenine). Dilute fresh YPD overnight culture 1/100
(e.g., 0.25 ml in 25 ml) and grow for ~5 hours (~2 doublings) to obtain OD600 0.2-0.3.
Plan to use ~1 OD unit per plasmid transformation and 3-5 OD units per linear DNA. For
example, 25 ml at OD600 = 0.2 is 5 OD units, which is enough for ~5 plasmids or 1-2 linear DNAs.
For best results: do steps 2-7 on ice with ice-cold solutions; microfuge 1 minute at 4000 rpm
(1500 g); heat denature and snap cool carrier DNA prior to use.
2. Chill cells on ice. Centrifuge in 50 ml tube for 5 minutes at 2000 rpm (~1000 g). Remove as much
supernatant as possible. Use an aspirator with a sterile tip (do this for all wash steps below).
3. Resuspend pellet in 1 ml sterile dH2O. Transfer to microfuge tube. Spin 30 seconds at top speed.
4. Wash cells twice with 1 ml sterile dH2O.
5. Resuspend pellet in 1 ml TE/LiOAc. Spin 30 seconds at top speed. Keep on ice for steps 5 - 7.
Make TE/LiOAc fresh and store on ice: 200 l 10X TE
200 l 1M LiOAc
1.6 ml sterile dH2O
6. Resuspend pellet in TE/LiOAc by gentle re-pipetting. Use 100 l per 1 OD600 unit for plasmid
DNA transformations. Use 100 l per 3-5 OD600 units for linear DNA transformations.
7. In 2.0 ml microfuge tube mix: 5 l 10 mg/ml carrier DNA
0.5 - 5 l transforming DNA (10 ng - 1 g DNA)
100 l yeast cells, gently mix by repipetting.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine