Freimoser:Protocols:FastYeastTransformation: Difference between revisions

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== Protocol: Fast yeast transformation==
== Protocol: Fast yeast transformation==


#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. (3 or 4 average colonies at least)
#Add in the following order:
#Add in the following order:
##1-2 µl DNA-plasmid (up to 30-50 ul)
##1-2 µl DNA-plasmid (up to 30-50 µl)
##240 µl 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
#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 H<sub>2</sub>O and plate everything on corresponding medium
#spin down, resuspend in 100μl H<sub>2</sub>O and plate everything on corresponding medium


==BioCoder version==
Following is the Freimoser:Fast yeast transformation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]]
====Text Output====
[[Freimoser:Fast yeast transformation protocol]]
====Source Code====
[[Freimoser:Fast yeast transformation protocol - source code]]


== Material: ==
== Material: ==


*Plasmid: 100 ng - 1 µg
*Plasmid: 100 ng - 1 &micro;g
*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 &micro;l 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 3'350) solution, filter-sterilized
*1M Li-Acetate, autoclaved
*1M Li-Acetate, autoclaved


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*Gietz-1992 pmid=1561104
*Gietz-1992 pmid=1561104
</biblio>
</biblio>
[[Category:Protocol]] [[Category:Yeast]] [[Category:DNA]]

Latest revision as of 02:34, 16 December 2010

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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 or 4 average colonies at least)
  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

BioCoder version

Following is the Freimoser:Fast yeast transformation protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder(see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Freimoser:Fast yeast transformation protocol

Source Code

Freimoser:Fast yeast transformation protocol - source code

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. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425. DOI:10.1093/nar/20.6.1425 | PubMed ID:1561104 | HubMed [Gietz-1992]
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