Freimoser:Protocols:FastYeastTransformation

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== Protocol: Fast yeast transformation==
== Protocol: Fast yeast transformation==
-
#50ul carrier DNA
+
#50 µl carrier DNA
#scrap cells from plate
#scrap cells from plate
-
#1-2ul DNA-plasmid (up to 30-50 ul)
+
#1-2 µl DNA-plasmid (up to 30-50 ul)
-
#240 ul PEG (50%)
+
#240 µl PEG (50%)
-
#36 ul 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-1ug
+
*Plasmid: 100 ng - 1 µg
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*carrier DNA: salmon/herring sperm DNA in H2O, 2mg/ml stock, 500 ul 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 33-50) solution, filter-sterilized
*50% poly-ethylen-glycol (PEG) (MW 33-50) solution, filter-sterilized
*1M Li-Acetate, autoclaved
*1M Li-Acetate, autoclaved

Revision as of 07:14, 17 October 2006


Protocol: Fast yeast transformation

  1. 50 µl carrier DNA
  2. scrap cells from plate
  3. 1-2 µl DNA-plasmid (up to 30-50 ul)
  4. 240 µl PEG (50%)
  5. 36 µl Li-Ac (1M)
  6. resuspend with blue tip
  7. incubate at 30°C or RT at least 30 min
  8. heat shock: 45°C, 15 min (or 42°C for 20 min)
  9. 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

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