High Efficency Yeast Plasmid Rescue
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High Efficency Yeast Plasmid Rescue
(by Trey Powers, Fields Lab )-
Grow yeast overnight.
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Spin down 1.5 ml of culture in microfuge tubes.
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Decant
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Add 250 µl of Qiagen buffer P1 to each tube
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Add about 250 µl of glass beads
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Beat or vortex on high for 3-5 minutes
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Add 250 µl of buffer P2 and gently mix well (Do not let lysis reaction continue for more than five minutes)
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Add 350 µl of buffer N3 and mix immediately, but gently
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Centrifuge for 10 minutes on full blast
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Apply supernatants to QIAprep spin column
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Centrifuge column for 45 seconds on full blast. Discard flow-though
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Wash column with 750 µl of Buffer PE and centrifuge 45 seconds
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Discard flow through and spin for an additional 45 seconds
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Place column in a clean tube and add 50 µl of buffer EB
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Let sit for 1 min. then spin for 1 min.
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For efficent transformation into coli use 1-2 µl of this prep per aliquot of E. coli using the 90sec heat shock protocol. (NOTE: using up to 20 µl will increase yield of transformants)
Colony Plasmid Rescue
Protocol adapted by Julia Sidorova and Linda Breeden from Hoffman and Winston, Gene , 1987, Vol. 57: 267-272.
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Start with a fresh plate of yeast, with large colonies grown for 3-4 days.
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Prepare eppendorfs with 20 µl aqueous buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).
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Pick colonies from plate with a pipette tip and resuspend in the aqueous buffer.
Note: Lab lore recommends NOT using a toothpick to pick your colony. There is something evil in toothpicks that inhibits bacterial transformations.
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Add 10 µl phenol and 10 µl chloroform (or 20 µl phenol:chloroform) to each eppendorf tube.
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Add 1/3 eppendorf cap (0.65 ml tube) of glass beads.
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Vortex 5 min. at speed of 4-5 in multihead vortexer at room temperature.
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Spin for 5 min. at maximum speed in a microcentrifuge.
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Thaw transformation competent E. coli cells on ice.
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In test tubes (for microfuge tubes see below; otherwise use 14-ml snap-cap tube) on ice add:
a) 1 µl aqueous phase (if you experience low yield, try using more)
b) 120 µl competent cells
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Mix and incubate on ice for 30 min.
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Heat shock for 90 sec at 42 deg C, then immediately add 2 ml of NZY broth to each test tube. (LB will also work, though richer media is usually better)
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Shake gently at 37 deg C for 1 hr. (If using microfuge tubes, rotate at 37°C in roller drum)
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Pellet cells for 5 min. at 3 krpm in table top centrifuge.
- Pour off the supernatant, resuspend the pellet in the residual liquid, and plate the entire suspension on selective medium.
