Difference between revisions of "Freimoser:Protocols:FastYeastTransformation"

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#incubate at 30°C or RT at least 30 min
 
#incubate at 30°C or RT at least 30 min
 
#heat shock: 45°C, 15 min (or 42°C for 20 min)
 
#heat shock: 45°C, 15 min (or 42°C for 20 min)
#spin down, resuspend in 100ul H2O and plate everything on corresponding medium
+
#spin down, resuspend in 100ul H<sub>2</sub>O and plate everything on corresponding medium
 +
 
  
 
== Material: ==
 
== Material: ==

Revision as of 13:54, 23 October 2006

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Protocol: Fast yeast transformation

  1. Add 50 µl carrier DNA to a 1.5 ml tube.
  2. scrap cells from plate and add to the carrier DNA.
  3. Add in the following order:
    1. 1-2 µl DNA-plasmid (up to 30-50 ul)
    2. 240 µl PEG (50%)
    3. 36 µl Li-Ac (1M)
  4. resuspend with blue tip
  5. incubate at 30°C or RT at least 30 min
  6. heat shock: 45°C, 15 min (or 42°C for 20 min)
  7. spin down, resuspend in 100ul H2O and plate everything on corresponding medium


Material:

  • Plasmid: 100 ng - 1 µg
  • carrier DNA: salmon/herring sperm DNA in H2O, 2mg/ml stock, 500 µl aliquots, heat-inactivated at 95°C for 5-6 min and put on ice before use (has to be done once).
  • 50% poly-ethylen-glycol (PEG) (MW 33-50) solution, filter-sterilized
  • 1M Li-Acetate, autoclaved


Reference

  1. Gietz D, St Jean A, Woods RA, and Schiestl RH. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425. PubMed ID:1561104 | HubMed [Gietz-1992]