Smolke:Protocols/Quick yeast transformation

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(New page: {{Smolke_Top}} ==Overview== Adapted from Gietz, Nature Protocols 2007 (dx.doiorg/10.1038/nprot.2007.14), this works similarly to the standard, longer method. -Leo ==Materials== *YPD *Ste...)
(Day 0)
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#Grow an overnight culture of your chosen yeast strain in appropriate media.  
#Grow an overnight culture of your chosen yeast strain in appropriate media.  
#*Alternatively, you can grow serial dilutions of your chosen strain, and one of them will likely be at the appropriate OD the next morning. For me, picking some cells into 3 mL of media, then taking 300 ul of this into a 25 mL flask works pretty well. -Leo
#*Alternatively, you can grow serial dilutions of your chosen strain, and one of them will likely be at the appropriate OD the next morning. For me, picking some cells into 3 mL of media, then taking 300 ul of this into a 25 mL flask works pretty well. -Leo
 +
#*Or you can pick ~10ul plated culture per transformation and resuspend in water. Ideally ~1e8 cells/transformation (can check OD). More variable but generally works. -Leo
===Day 1===
===Day 1===

Revision as of 13:20, 19 June 2013

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Contents

Overview

Adapted from Gietz, Nature Protocols 2007 (dx.doiorg/10.1038/nprot.2007.14), this works similarly to the standard, longer method. -Leo

Materials

  • YPD
  • Sterile water
  • 1M Lithium Acetate
  • 50% PEG (4000)
  • Salmon sperm DNA ('SSD', 10 mg/mL, stored at -20C)
  • Plasmid DNA

Procedure

Day 0

  1. Grow an overnight culture of your chosen yeast strain in appropriate media.
    • Alternatively, you can grow serial dilutions of your chosen strain, and one of them will likely be at the appropriate OD the next morning. For me, picking some cells into 3 mL of media, then taking 300 ul of this into a 25 mL flask works pretty well. -Leo
    • Or you can pick ~10ul plated culture per transformation and resuspend in water. Ideally ~1e8 cells/transformation (can check OD). More variable but generally works. -Leo

Day 1

  1. Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice.
  2. Pellet cells at ~6000g for 1 minute. Discard supernatant
  3. Add to the pellet the following, in order, then quickly vortex. If doing multiple transformations, you can make a master mix of everything except the DNA and aliquot.
    • 240 ul 50% PEG
    • 36 ul 1M LiAc
    • 10 ul single-stranded carrier DNA (10 mg/ml)
    • 74 ul of sterile water plus any plasmid DNA
  4. Incubate in 42°C water bath 15-40 minutes
  5. Spin 30 seconds at max speed and carefully remove supernatant
  6. Resuspend in 100-200 ul sterile water by vortexing and plate on appropriate plates.
  7. Let grow at 30°C 2-3 days


Day 3

Colonies should be ready. Some strains/plasmids may take an extra day.

Notes

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