Overview
Colony PCR from yeast, either to check inserts etc. or for sequencing.
Josh's Version (NaOH lysis)
Materials
Procedure
- Aliquot 20uL NaOH into PCR tubes
- Pick colonies (I use pipet tips) into the NaOH
- Incubate at 95C for ~45 minutes
- Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge)
- Use 1uL of supernatant as template in a (10uL) PCR.
Yeast PCR Lyticase lysis buffer
Lyticase buffer
(Kwiatkowski et al, 1990)
- 22.5 uL Tween 20 (= polyoxyethylene (20) sorbitan monolaurate)
- 22.5 uL NP-40 (= Igepal CA-630)
- 83.3 uL 3 M KCl
- 50 uL 1 M Tris HCl pH 8.3
- 150 uL 50 mM MgCl2
- 5 mg Lyticase (cat number L4025-250KU)
To 5 mL total volume with millipore water (4.67 mL).
Aliquot and store at -20 °C.
Procedure
Pick a small amount of each colony into 15 uL lysis buffer, suspend.
Heat in PCR block 60 min at 37 °C then 10 min at 95 °C. Pellet in microcentrifuge at max speed for 2 min. Use 2 ul/20ul PCR reaction.
Travis' Version (Lyticase lysis)
Materials
- Lyticase (from Sigma)
- TE
- PCR buffers, primers, polymerase, etc.
Procedure
The basic idea is breaking the cells with lyticase and heat, then doing PCR.
- Dilute stock of lyticase to 50 U/mL in TE.
- Aliquot lyticase in 50uL quantities
- Pick colonies (I use a pipette tip) and add to lyticase aliquots, pipette up and down or agitate to break up colony
- Incubate at 37°C for 30 min
- Incubate at 95°C for 10 min
- Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction
Notes
- I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards.
- The amount of yeast doesn't seem to matter much. I get success with very small colonies or with loads of cells.
Genomic Prep by Harju Bust'n'Grab
Materials
- overnight yeast cultures
- lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)
- phenol:chloroform
- chloroform
- ethanol (70% and 100%)
- sodium acetate
- TE
Procedure
Adapted by Kate from Harju et al., 2004
- Spin down 1.5 ml O/N culture and remove media
- Re-suspend in 200 µl lysis buffer and transfer to a thin-walled PCR tube
- Freeze at -80 °C, 5 min
- Thaw at 95 °C, 1 min on PCR block
- Repeat once and transfer to 1.5 mL eppendorf tube
- Vortex vigorously
- Add 200 µl phenol:chloroform and vortex well
- Spin 2 min, remove aqueous phase
- Add 200 µl chloroform and vortex well
- Spin 2 min, remove aqueous phase
- Add 400 µl ice-cold ethanol and 40 µl sodium acetate
- Precipitate at -80 °C, 5 min
- Spin 5 min at 4 °C
- Wash pellet with 70% ethanol
- Re-suspend in 50 µl TE - use 1 µl in a PCR reaction
Genomic Prep with Glass Beads
Materials
- overnight yeast cultures
- lysis buffer - 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA
- phenol:chloroform:isoamyl alcohol
- ice cold 100% EtOH
- glass beads
Procedure
Adapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie
- Grow 3-10 ml culture of yeast to saturation. (You can even use this protocol on single colonies if you don't need much DNA.)
- Collect cells by centrifugation in 1.5 ml eppendorf tube (max speed, 15 s).
- Aspirate supernatant and resuspend in 500 µl of ddH2O. Pellet, decant.
- Pipette briefly to resuspend pellet in residual liquid.
- Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads.
- Tape to vortex machine in cold room, vortex for 10-20 min. (You can vortex for 2 min at room temp if you don't need much DNA.)
- Add 200 ul PCI (phenol:chloroform:isoamyl alcohol; 25:24:1), vortex briefly.
- Centrifuge at max for 5 min (4 °C).
- Transfer aqueous top layer to fresh eppendorf tube.
- Add 50 ul 3M sodium acetate, then 1 ml ice cold 100% EtOH.
- Spin for 10-20 min at high speed at 4 °C.
- Decant off liquid and vacufuge until pellet dries.
- Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer.
- For PCR use 1 ul of 1:10 or 1:100 dilution of the resuspension as template - this varies depending on the strain, shearing, and how well your genomic isolation went. You can nanodrop the resuspension to estimate the concentration, and use 10-30 ng in a 25 uL PCR reaction.
Notes
Yeast colony PCR works best on freshly grown colonies, immediately after transformation.
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