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 | #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 | #Repellet. Resuspend in 200 μL sterile water per transformation. | ||
#Repellet. Resuspend in | #Repellet. Resuspend in 200 μL LiAc/TE per transformation. | ||
#Repellet. Resuspend in | #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. | #Add DNA to each tube. 1 μL of a prep is usually more than necessary. | ||
#Add | #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 | #Incubate in the spinner at 30 °C for 30 minutes. | ||
#Heat shock in | #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 | #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. | ||
# | #Gently resuspend in 100 μL sterile water, plate on appropriate dropout plates. | ||
===Day 3=== | ===Day 3=== | ||
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==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, 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
Materials
ProcedureDay 0Grow an overnight culture of your chosen yeast strain in appropriate media Day 1
Day 3Colonies should be ready. Some strains/plasmids may take an extra day. NotesI 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 |