Smolke:Protocols/Yeast Colony PCR: Difference between revisions
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#Centrifuge at max speed for 10 minutes (helps to use plastic inserts in the microfuge) | #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. | #Use 1uL of supernatant as template in a (10uL) PCR. | ||
==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. |
Revision as of 11:58, 6 December 2011
OverviewColony PCR from yeast, either to check inserts etc. or for sequencing. Travis' Version (Lyticase lysis)Materials
ProcedureThe basic idea is breaking the cells with lyticase and heat, then doing PCR.
Notes
Josh's Version (NaOH lysis)Materials
Procedure
Genomic Prep by Harju Bust'n'GrabMaterials
ProcedureAdapted by Kate from Harju et al., 2004
Genomic Prep with Glass BeadsMaterials
ProcedureAdapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie
NotesYeast colony PCR works best on freshly grown colonies, immediately after transformation. |