Freimoser:Protocols:FastYeastTransformation

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== Protocol: Fast yeast transformation==
== Protocol: Fast yeast transformation==
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#Add 50 μl carrier DNA to a 1.5 ml tube.
+
#Add 50 µl carrier DNA to a 1.5 ml tube.
#scrap cells from plate and add to the carrier DNA.
#scrap cells from plate and add to the carrier DNA.
#Add in the following order:
#Add in the following order:
-
##1-2 μl DNA-plasmid (up to 30-50 μl)
+
##1-2 µl DNA-plasmid (up to 30-50 µl)
-
##240 vl PEG (50%)
+
##240 µl PEG (50%)
-
##36 μl Li-Ac (1M)
+
##36 µl Li-Ac (1M)
#resuspend with blue tip
#resuspend with blue tip
#incubate at 30°C or RT at least 30 min
#incubate at 30°C or RT at least 30 min
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== Material: ==
== Material: ==
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*Plasmid: 100 ng - 1 μg
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*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).
*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 3'350) solution, filter-sterilized
*50% poly-ethylen-glycol (PEG) (MW 3'350) solution, filter-sterilized

Revision as of 06:56, 22 December 2006


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 µl)
    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 100μl 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 3'350) solution, filter-sterilized
  • 1M Li-Acetate, autoclaved


Reference

  1. Gietz D, St Jean A, Woods RA, and Schiestl RH. . pmid:1561104. PubMed HubMed [Gietz-1992]
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