McClean:Making and Using Frozen Yeast Competant Cells: Difference between revisions

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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.
# 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.
* Pre-warm 500 mL YPD in the 2 L flask at 30°C for later use.  
##Pre-warm 500 mL YPD in the 2 L flask at 30°C for later use.  
# Take this culture and find the OD600 to determine cell count (OD 0.1 ≈ 1 x 10^6 cells/mL.) An OD close to 1 is desired.
# Take this culture and find the OD600 to determine cell count (OD 0.1 ≈ 1 x 10^6 cells/mL.) An OD close to 1 is desired.
# With this culture, innoculate the pre-warmed flask of YPD with the entire 25mL and shake at 30°C for about 4 hours
# With this culture, innoculate the pre-warmed flask of YPD with the entire 25mL and shake at 30°C for about 4 hours
* NOTE that adding cells according to their titers as described in Geitz et. al will not be possible since the cell densities achieved were not even close to what was described (try using 2X YPD if this is the route you need to go in.)
##NOTE that adding cells according to their titers as described in Geitz et. al will not be possible since the cell densities achieved were not even close to what was described (try using 2X YPD if this is the route you need to go in.)
# Harvest the cells by centrifugation at 3,000xg for 5 minutes using 50 mL conical tubes (since this is what we happen to have in spades.) I split 250 mL of the culture among 5 tubes, spin them down, discard the supernatant, and add the remaining culture to the same tubes and repeat.
# Harvest the cells by centrifugation at 3,000xg for 5 minutes using 50 mL conical tubes (since this is what we happen to have in spades.) I split 250 mL of the culture among 5 tubes, spin them down, discard the supernatant, and add the remaining culture to the same tubes and repeat.
# Wash the cells in 0.5 volumes (25 mL in each of these tubes) of  sterile water. Resuspend in 0.01 volumes (500μL each) sterile water and transfer to microfuge tubes. Pellet cells at 3,000xg for 5 minutes.
# Wash the cells in 0.5 volumes (25 mL in each of these tubes) of  sterile water. Resuspend in 0.01 volumes (500μL each) sterile water and transfer to microfuge tubes. Pellet cells at 3,000xg for 5 minutes.

Latest revision as of 11:58, 29 October 2013

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 'microfuge' 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.
    1. Pre-warm 500 mL YPD in the 2 L flask at 30°C for later use.
  2. Take this culture and find the OD600 to determine cell count (OD 0.1 ≈ 1 x 10^6 cells/mL.) An OD close to 1 is desired.
  3. With this culture, innoculate the pre-warmed flask of YPD with the entire 25mL and shake at 30°C for about 4 hours
    1. NOTE that adding cells according to their titers as described in Geitz et. al will not be possible since the cell densities achieved were not even close to what was described (try using 2X YPD if this is the route you need to go in.)
  4. Harvest the cells by centrifugation at 3,000xg for 5 minutes using 50 mL conical tubes (since this is what we happen to have in spades.) I split 250 mL of the culture among 5 tubes, spin them down, discard the supernatant, and add the remaining culture to the same tubes and repeat.
  5. Wash the cells in 0.5 volumes (25 mL in each of these tubes) of sterile water. Resuspend in 0.01 volumes (500μL each) sterile water and transfer to microfuge tubes. Pellet cells at 3,000xg for 5 minutes.
  6. Resuspend in 500μL FCC solution and aliquot 50μL each into microfuge tubes.
  7. Place these tubes into the styrofoam racks and store at -80°C. Since the racks are to promote slow freezing, after the tubes are brought to -80°C you may transfer them to whatever your container of choice is in the -80.

Using These Cells for Transformation

Take out a tube for each set of transformations and warm between your hands for 15 to 30 seconds. Pellet the cells at 3,000xg for 2 minutes and discard the supernatant. Take these cells and add them to your transformation master mix and follow the procedure as described in McClean:Yeast Transformation day 2 step 7. You may need to use more than one tube of cells if your yield was low.

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.H. Schiestl. (2002) Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nature Prot. Vol. 2 No. 1.

Contact

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