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
#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===

Revision as of 10:09, 23 May 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
  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, 0.3mg/mL 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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