Smolke:Protocols/Yeast Colony PCR: Difference between revisions
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==Overview== | ==Overview== | ||
Colony PCR from yeast, either to check inserts etc. or for sequencing. | Colony PCR from yeast, either to check inserts etc. or for sequencing. | ||
==Josh's Version (NaOH lysis)== | |||
===Materials=== | |||
*20 mM NaOH | |||
*PCR 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)== | ==Travis' Version (Lyticase lysis)== | ||
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*I use this protocol to PCR off the chromosome for sequencing... I clean up the rxn with a Zymoclean kit afterwards. | *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. | *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== | ==Genomic Prep by Harju Bust'n'Grab== | ||
===Materials=== | ===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=== | ===Procedure=== | ||
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#Re-suspend in 50 µl TE - use 1 µl in a PCR reaction | #Re-suspend in 50 µl TE - use 1 µl in a PCR reaction | ||
==Genomic Prep with Glass Beads== | |||
==Genomic Prep | |||
===Materials=== | ===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=== | ===Procedure=== | ||
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#Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads. | #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.) | #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 | #Add 200 ul PCI (phenol:chloroform:isoamyl alcohol; 25:24:1), vortex briefly. | ||
#Centrifuge at max for 5 min (4 °C). | #Centrifuge at max for 5 min (4 °C). | ||
#Transfer aqueous top layer to fresh eppendorf tube. | #Transfer aqueous top layer to fresh eppendorf tube. | ||
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#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. | #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. |
Latest revision as of 15:01, 22 May 2013
OverviewColony PCR from yeast, either to check inserts etc. or for sequencing. Josh's Version (NaOH lysis)Materials
Procedure
Yeast PCR Lyticase lysis bufferLyticase buffer(Kwiatkowski et al, 1990)
To 5 mL total volume with millipore water (4.67 mL). Aliquot and store at -20 °C. ProcedurePick 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
ProcedureThe basic idea is breaking the cells with lyticase and heat, then doing PCR.
Notes
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. |