Smolke:Protocols/Yeast transformation: Difference between revisions

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#*If you're transforming a plasmid, you don't need high efficiency. Just pull the culture whenever it's convenient.
#*If you're transforming a plasmid, you don't need high efficiency. Just pull the culture whenever it's convenient.
#Prepare your solutions - put the water, LiAc/TE, and 40% PEG on ice. Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice.
#Prepare your solutions - put the water, LiAc/TE, and 40% PEG on ice. Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice.
#Pellet cells at 6000g for 5 minutes at 4C. Resuspend in 2mL sterile water per transformation (so 10mL water for 5 transformations).
#Pellet cells at 3-6000g for 5 minutes at 20 °C. Resuspend in 2 mL sterile water per transformation (so 10 mL water for 5 transformations).
#Repellet. Resuspend in 200uL sterile water per transformation.
#Repellet. Resuspend in 200 μL sterile water per transformation.
#Repellet. Resuspend in 200uL LiAc/TE per transformation.
#Repellet. Resuspend in 200 μL LiAc/TE per transformation.
#Repellet. Resuspend in 50uL LiAc/TE per transformation. Add 5uL SSD per transformation. Aliquot into chilled eppendorf tubes, 55uL per tube.
#Repellet. Resuspend in 50 μL LiAc/TE per transformation. Add 5 μL SSD per transformation. Aliquot into chilled eppendorf tubes, 55 μL per tube.
#Add DNA to each tube. 1uL of a prep is usually more than necessary.
#Add DNA to each tube. 1 μL of a prep is usually more than necessary.
#Add 300uL 40% PEG to each tube. Pipet to resuspend (shouldn't see any streaks after resuspension)
#Add 300 μL 40% PEG to each tube. Pipet or vortex to resuspend (shouldn't see any streaks after resuspension).
#Incubate in the spinner at 30C for 30 minutes  
#Incubate in the spinner at 30 °C for 30 minutes.
#Heat shock in 42C water bath for 15 minutes
#Heat shock in 42 °C water bath for 15 minutes (can be lengthened up to 40 minutes).
#Spin down, remove PEG
#Spin down, remove PEG.
#If using G418, resuspend in 1mL YPD, incubate at 30C for ~2hr, then repellet
#If using G418, resuspend in 1 mL YPD, incubate at 30C for 2-3 hr, then repellet. Alternatively, plate on YPD and let recover overnight, then use replicate plater to replate on selective plates the next day.
#Resuspend in 100uL sterile water, plate on appropriate dropout plates
#Gently resuspend in 100 μL sterile water, plate on appropriate dropout plates.


===Day 3===
===Day 3===
Line 35: Line 35:


==Notes==
==Notes==
I get higher efficiency if I dilute the overnight culture to OD 0.2-0.4 and heat shock for 25 min instead of 15 min.  (Gietz & Schiestl's 2007 Nature Protocols paper recommends up to 40 min heat shock, optimized for your strain.)  Adjust amounts of PEG (50% w/v), LiAc, and ssDNA so that each transformation has about 10<math>^8</math> cells, 35%w/v PEG, 0.1M LiAc, 0.3mg/mL ssDNA.  Cells can be pelleted at 20 °C.  Some transformations take 3-4 days for colonies to grow. ~Stephanie
I get higher efficiency if I dilute the overnight culture to OD 0.2-0.4 and heat shock for 25 min instead of 15 min.  (Gietz & Schiestl's 2007 Nature Protocols paper recommends up to 40 min heat shock, optimized for your strain.)  Adjust amounts of PEG (50% w/v), LiAc, and ssDNA so that each transformation has about 10<math>^8</math> cells, 35%w/v PEG, 0.1M LiAc, and 10 μg ssDNA.  Cells can be pelleted at 20 °C.  Some transformations take 3-4 days for colonies to grow. See http://labs.fhcrc.org/gottschling/Yeast%20Protocols/ytrans.html for more possible variations. ~Stephanie G

Latest revision as of 14:50, 29 November 2011

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Materials

  • YPD
  • Sterile water
  • Lithium Acetate/TE: 0.1M LiAc, 10mM Tris, 1mM EDTA, pH 7.5 (Mix 1mL 10x LiAc + 1mL 10x TE + 8mL sterile water)
  • 40% PEG: 40% PEG, 0.1M LiAc, 10mM Tris, 1mM EDTA, pH 7.5 (Mix 1mL 10x LiAc + 1mL 10x TE + 8mL 50% PEG)
    • I've seen protocols use different molecular weights of PEG. 3000-4000 seems pretty common
  • Salmon sperm DNA ('SSD', 10 ug/uL, stored at -20C)
  • Plasmid DNA

Procedure

Day 0

Grow an overnight culture of your chosen yeast strain in appropriate media

Day 1

  1. In the morning, dilute overnight culture 1:100 (~OD 0.1) into fresh media. Use 5mL fresh media per transformation.
  2. Grow the culture to OD 0.4-0.8 (3.5-4.5 hr)
    • If you're transforming a plasmid, you don't need high efficiency. Just pull the culture whenever it's convenient.
  3. Prepare your solutions - put the water, LiAc/TE, and 40% PEG on ice. Thaw the SSD, make an aliquot into a PCR tube, incubate it at 95C for ~5 minutes, then chill on ice.
  4. Pellet cells at 3-6000g for 5 minutes at 20 °C. Resuspend in 2 mL sterile water per transformation (so 10 mL water for 5 transformations).
  5. Repellet. Resuspend in 200 μL sterile water per transformation.
  6. Repellet. Resuspend in 200 μL LiAc/TE per transformation.
  7. Repellet. Resuspend in 50 μL LiAc/TE per transformation. Add 5 μL SSD per transformation. Aliquot into chilled eppendorf tubes, 55 μL per tube.
  8. Add DNA to each tube. 1 μL of a prep is usually more than necessary.
  9. Add 300 μL 40% PEG to each tube. Pipet or vortex to resuspend (shouldn't see any streaks after resuspension).
  10. Incubate in the spinner at 30 °C for 30 minutes.
  11. Heat shock in 42 °C water bath for 15 minutes (can be lengthened up to 40 minutes).
  12. Spin down, remove PEG.
  13. If using G418, resuspend in 1 mL YPD, incubate at 30C for 2-3 hr, then repellet. Alternatively, plate on YPD and let recover overnight, then use replicate plater to replate on selective plates the next day.
  14. Gently resuspend in 100 μL sterile water, plate on appropriate dropout plates.

Day 3

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

Notes

I get higher efficiency if I dilute the overnight culture to OD 0.2-0.4 and heat shock for 25 min instead of 15 min. (Gietz & Schiestl's 2007 Nature Protocols paper recommends up to 40 min heat shock, optimized for your strain.) Adjust amounts of PEG (50% w/v), LiAc, and ssDNA so that each transformation has about 10[math]\displaystyle{ ^8 }[/math] cells, 35%w/v PEG, 0.1M LiAc, and 10 μg ssDNA. Cells can be pelleted at 20 °C. Some transformations take 3-4 days for colonies to grow. See http://labs.fhcrc.org/gottschling/Yeast%20Protocols/ytrans.html for more possible variations. ~Stephanie G

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