McClean:Making and Using Frozen Yeast Competant Cells

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'''Frozen Competant Cell (FCC) Solution '''
'''Frozen Competant Cell (FCC) Solution '''
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Solution is 5% (v/v) glycerol and 10% (v/v)DMSO. Be sure to filter-sterilize.  
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Solution is 5% (v/v) glycerol and 10% (v/v) DMSO. Be sure to filter-sterilize.  
==Cell Preparation==
==Cell Preparation==
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# Innoculate an overnight culture of your strain into 25 mL of YPD and grow at 30°C and 200 RPM overnight or for 12-16 hours.
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#
==Notes==
==Notes==

Revision as of 14:11, 29 October 2013

Contents

Overview

The following is a protocol to freeze down and use any of our S. cerevisae strains for standard LiOAc transformation at a later date. This is especially useful when you often need to perform many transformations using a single background strain. Adapted from the protocol by Geitz et. al 2007, Nature Protocols.

Materials

  • 50 mL Conical Tubes
  • Frozen Competant Cell Solution (recipe to follow)
  • Liquid YPD media (From media facility, or recipe in Amberg et. al's Methods in Yeast Genetics)
  • 100 well styrofoam freezer racks (that will fit standard 1.5 mL 'minifuge' tubes
  • A 250 mL sterile culture flask for each strain
  • A 2 L sterile flask for each strain


Stock Solutions

Frozen Competant Cell (FCC) Solution

Solution is 5% (v/v) glycerol and 10% (v/v) DMSO. Be sure to filter-sterilize.

Cell Preparation

  1. Innoculate an overnight culture of your strain into 25 mL of YPD and grow at 30°C and 200 RPM overnight or for 12-16 hours.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.

Contact

or instead, discuss this protocol.

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