Smolke:Protocols/Yeast Colony PCR - Revision history

Stephanie Galanie at 18:58, 6 December 2011

2011-12-06T18:58:06Z

←Older revision		Revision as of 18:58, 6 December 2011
Line 89:		Line 89:
	#Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer.		#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.		#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.

Stephanie Galanie: /* Genomic Prep by Phenol:Chloroform with Glass Beads */

2011-10-27T05:00:30Z

Genomic Prep by Phenol:Chloroform with Glass Beads

←Older revision		Revision as of 05:00, 27 October 2011
Line 65:		Line 65:
	#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 ~~by Phenol:Chloroform~~ with Glass Beads==	+	==Genomic Prep with Glass Beads==
	===Materials===		===Materials===
	*overnight yeast cultures		*overnight yeast cultures

Stephanie Galanie: /* Genomic Prep by Phenol:Chloroform with Glass Beads */

2011-10-27T04:59:22Z

Genomic Prep by Phenol:Chloroform with Glass Beads

←Older revision		Revision as of 04:59, 27 October 2011
Line 67:		Line 67:
	==Genomic Prep by Phenol:Chloroform with Glass Beads==		==Genomic Prep by Phenol:Chloroform with Glass Beads==
	===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===
Line 77:		Line 81:
	#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~~:TE~~; 25:24:1), vortex briefly.	+	#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.
Line 85:		Line 89:
	#Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer.		#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.		#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.
-
-	~~Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA.~~

Stephanie Galanie: /* Genomic Prep by Harju Bust'n'Grab */

2011-10-27T04:57:36Z

Genomic Prep by Harju Bust'n'Grab

←Older revision		Revision as of 04:57, 27 October 2011
Line 38:		Line 38:
	==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===
Line 58:		Line 64:
	#Wash pellet with 70% ethanol		#Wash pellet with 70% ethanol
	#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
-
-	~~Note: Lysis buffer contains 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)~~

	==Genomic Prep by Phenol:Chloroform with Glass Beads==		==Genomic Prep by Phenol:Chloroform with Glass Beads==

Stephanie Galanie: /* Procedure */

2011-10-27T04:55:34Z

Procedure

←Older revision		Revision as of 04:55, 27 October 2011
Line 66:		Line 66:

	===Procedure===		===Procedure===
-	Adapted by Mike from protocol from friend in the Herschlag group:	+	Adapted by Mike from protocol from friend in the Herschlag group, and further adapted by Stephanie
-	1. Grow 5-10 ml culture of yeast to saturation.	+	#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.)
-	2. Collect cells by centrifugation in 15 ml ~~conical~~ tube (~~~3000xg~~, ~~5-10 min~~).	+	#Collect cells by centrifugation in 1.5 ml eppendorf tube (max speed, 15 s).
-	3. Aspirate supernatant and resuspend in 500 µl of ddH2O.	+	#Aspirate supernatant and resuspend in 500 µl of ddH2O. Pellet, decant.
-	~~4. Transfer cells to 1.5 ml eppendorf tube~~.	+	#Pipette briefly to resuspend pellet in residual liquid.
-	~~5. Centrifuge at 3500rcf at room temp for 3.5 min.~~	+	#Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads.
-	~~6. Decant.~~	+	#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.)
-	7. Pipette briefly to resuspend pellet in residual liquid.	+	#Add 200 ul PCI (phenol:chloroform:isoamyl alcohol:TE; 25:24:1), vortex briefly.
-	8. Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads.	+	#Centrifuge at max for 5 min (4 °C).
-	9. Tape to vortex machine in cold room, vortex for 10-~~20min~~.	+	#Transfer aqueous top layer to fresh eppendorf tube.
-	10. Add 200 ul PCI (phenol:chloroform:isoamyl alcohol:TE; 25:24:1), vortex briefly.	+	#Add 50 ul 3M sodium acetate, then 1 ml ice cold 100% EtOH.
-	~~11.~~ Centrifuge at max for 5 min (4C).	+	#Spin for 10-20 min at high speed at 4 °C.
-	~~12.~~ Transfer aqueous top layer to fresh eppendorf tube.	+	#Decant off liquid and vacufuge until pellet dries.
-	~~13.~~ Add 1 ml ice cold 100% EtOH ~~+ 50 ul 3M sodium acetate~~.	+	#Resuspend in ~100 uL water, 1x TE buffer pH 8.0, or EB buffer.
-	~~14.~~ Spin for 10-~~20min~~ high speed ~~@ 4C~~.	+	#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.
-	~~15.~~ Decant off liquid and vacufuge until pellet dries.	+
-	~~16.~~ Resuspend in water, 1x TE buffer pH 8.0, or EB buffer ~~~100ul~~	+
-	~~17. Test variety of dilutions for~~ PCR (1 ul of 1:10 or 1:100 as template ~~for PCR often give good results) – unfortunately,~~ this ~~is somewhat empirical based upon~~ the strain, shearing, and how well your genomic isolation went.	+

	Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA.		Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA.

Stephanie Galanie: /* Procedure */

2011-10-27T04:48:40Z

Procedure

←Older revision		Revision as of 04:48, 27 October 2011
Line 43:		Line 43:
	Adapted by Kate from Harju et al., 2004		Adapted by Kate from Harju et al., 2004

-	• Spin down 1.5 ml O/N culture and remove media	+	#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	+	#Re-suspend in 200 µl lysis buffer and transfer to a thin-walled PCR tube
-	• Freeze at -~~80oC~~, 5 min	+	#Freeze at -80 °C, 5 min
-	• Thaw at ~~95oC~~, 1 min on PCR block	+	#Thaw at 95 °C, 1 min on PCR block
-	• Repeat once and transfer to 1.5 mL eppendorf tube	+	#Repeat once and transfer to 1.5 mL eppendorf tube
-	• Vortex vigorously	+	#Vortex vigorously
-	• Add 200 µl phenol:chloroform and vortex well	+	#Add 200 µl phenol:chloroform and vortex well
-	• Spin 2 min, remove aqueous phase	+	#Spin 2 min, remove aqueous phase
-	• Add 200 µl chloroform and vortex well	+	#Add 200 µl chloroform and vortex well
-	• Spin 2 min, remove aqueous phase	+	#Spin 2 min, remove aqueous phase
-	• Add 400 µl ice-cold ethanol and 40 µl sodium acetate	+	#Add 400 µl ice-cold ethanol and 40 µl sodium acetate
-	• Precipitate at -~~80oc~~, 5 min	+	#Precipitate at -80 °C, 5 min
-	• Spin 5 min at ~~4oC~~	+	#Spin 5 min at 4 °C
-	• Wash pellet with 70% ethanol	+	#Wash pellet with 70% ethanol
-	• 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
-	~~Lysis buffer contains 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)~~	+

		+	Note: Lysis buffer contains 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)

	==Genomic Prep by Phenol:Chloroform with Glass Beads==		==Genomic Prep by Phenol:Chloroform with Glass Beads==

Stephanie Galanie at 04:44, 27 October 2011

2011-10-27T04:44:45Z

←Older revision		Revision as of 04:44, 27 October 2011
Line 35:		Line 35:
	#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===
		+
		+
		+	===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 -80oC, 5 min
		+	• Thaw at 95oC, 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 -80oc, 5 min
		+	• Spin 5 min at 4oC
		+	• Wash pellet with 70% ethanol
		+	• Re-suspend in 50 µl TE - use 1 µl in a PCR reaction
		+	Lysis buffer contains 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)
		+
		+
		+	==Genomic Prep by Phenol:Chloroform with Glass Beads==
		+	===Materials===
		+
		+
		+	===Procedure===
		+	Adapted by Mike from protocol from friend in the Herschlag group:
		+	1. Grow 5-10 ml culture of yeast to saturation.
		+	2. Collect cells by centrifugation in 15 ml conical tube (~3000xg, 5-10 min).
		+	3. Aspirate supernatant and resuspend in 500 µl of ddH2O.
		+	4. Transfer cells to 1.5 ml eppendorf tube.
		+	5. Centrifuge at 3500rcf at room temp for 3.5 min.
		+	6. Decant.
		+	7. Pipette briefly to resuspend pellet in residual liquid.
		+	8. Add 200 ul detergent lysis buffer, ~100 mg (50uL) glass beads.
		+	9. Tape to vortex machine in cold room, vortex for 10-20min.
		+	10. Add 200 ul PCI (phenol:chloroform:isoamyl alcohol:TE; 25:24:1), vortex briefly.
		+	11. Centrifuge at max for 5 min (4C).
		+	12. Transfer aqueous top layer to fresh eppendorf tube.
		+	13. Add 1 ml ice cold 100% EtOH + 50 ul 3M sodium acetate.
		+	14. Spin for 10-20min high speed @ 4C.
		+	15. Decant off liquid and vacufuge until pellet dries.
		+	16. Resuspend in water, 1x TE buffer pH 8.0, or EB buffer ~100ul
		+	17. Test variety of dilutions for PCR (1 ul of 1:10 or 1:100 as template for PCR often give good results) – unfortunately, this is somewhat empirical based upon the strain, shearing, and how well your genomic isolation went.
		+
		+	Note: Detergent lysis buffer is 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 10 mM EDTA.

Stephanie Galanie: /* Josh's Version */

2011-10-27T04:40:38Z

Josh's Version

←Older revision		Revision as of 04:40, 27 October 2011
Line 24:		Line 24:
	*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.

-	==Josh's Version==	+	==Josh's Version (NaOH lysis)==
	===Materials===		===Materials===
	*20 mM NaOH		*20 mM NaOH

Stephanie Galanie: /* Travis' Version */

2011-10-27T04:40:22Z

Travis' Version

←Older revision		Revision as of 04:40, 27 October 2011
Line 4:		Line 4:
	Colony PCR from yeast, either to check inserts etc. or for sequencing.		Colony PCR from yeast, either to check inserts etc. or for sequencing.

-	==Travis' Version==	+	==Travis' Version (Lyticase lysis)==
	===Materials===		===Materials===
	*Lyticase (from Sigma)		*Lyticase (from Sigma)

Josh K. Michener at 21:12, 20 October 2009

2009-10-20T21:12:47Z

←Older revision		Revision as of 21:12, 20 October 2009
Line 2:		Line 2:

	==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.

-	==~~Materials~~==	+	==Travis' Version==
-		+	===Materials===
	*Lyticase (from Sigma)		*Lyticase (from Sigma)
	*TE		*TE
	*PCR buffers, primers, polymerase, etc.		*PCR buffers, primers, polymerase, etc.

-	==Procedure==	+	===Procedure===
	The basic idea is breaking the cells with lyticase and heat, then doing PCR.		The basic idea is breaking the cells with lyticase and heat, then doing PCR.
-

	#Dilute stock of lyticase to 50 U/mL in TE.		#Dilute stock of lyticase to 50 U/mL in TE.
Line 22:		Line 20:
	#Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction		#Use as template for PCR - I use 5uL of the cells in a 50uL PCR reaction

-		+	===Notes===
-	==Notes==	+
	*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.

-	==~~Contact~~==	+	==Josh's Version==
-	~~[[User:Tsbayer\|Travis]]~~	+	===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.