Davebrid: formatting changes

2009-12-14T15:02:28Z

formatting changes

Davebrid: initial copy from protocol folder

2009-12-14T15:01:21Z

initial copy from protocol folder

New page

Materials
• YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask
• TE/LiAc prepare fresh by combining 150 uL 10X TE, 150 uL 1M LiAc and 1.2 mL water
• PEG/LiAc prepare fresh by adding 8 mL 50% PEG-3350 to 1 mL 10X TE and 1 mL 1M LiAc in a 15 mL falcon tube
• Herring testes carrier DNA. Boil 20min then place on ice before use
Protocol
1. Grow 50mL overnight culture of yeast in YPDA or SD. Add several colonies to 1mL vortexing to break up clumps then pouring into 50 mL media in a 250 mL Erlenmeyer flask. Grow at 30C
2. Transfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3
3. Incubate 3h at 30C to an OD of 0.4-0.6
4. Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.
5. Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min
6. Discard supernatant and resuspend in 50 mL sterile TE or water
7. Centrifuge 1000g for 5min
8. Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations
9. For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)
10. Add 100 uL yeast cells and mix by vortexing
11. Add 0.6 mL PEG/LiAc solution and vortex to mix
12. Incubate at 30C for 30min with shaking
13. Add 70 uL DMSO
14. Heat shock for 15min at 42C swirling occasionally to mix
15. Chill on ice for 1-2 min
16. Centrifuge at high speed for 5 s to pellet cells
17. Resuspend in 0.5mL TE
18. Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media
19. Allow cells to grow for 3-5 days on selective media.

[[Category:Yeast]]
[[Category:Molecular Biology]]
[[Category:Transfection]]

← Older revision		Revision as of 15:02, 14 December 2009
Line 1:		Line 1:
	Materials		==Materials==
	• YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask		*YPDA Media 50 mL in a 250 mL flask and 300 mL in a 1L flask
	• TE/LiAc prepare fresh by combining 150 uL 10X TE, 150 uL 1M LiAc and 1.2 mL water		*TE/LiAc prepare fresh by combining 150 uL 10X TE, 150 uL 1M LiAc and 1.2 mL water
	• PEG/LiAc prepare fresh by adding 8 mL 50% PEG-3350 to 1 mL 10X TE and 1 mL 1M LiAc in a 15 mL falcon tube		*PEG/LiAc prepare fresh by adding 8 mL 50% PEG-3350 to 1 mL 10X TE and 1 mL 1M LiAc in a 15 mL falcon tube
	• Herring testes carrier DNA. Boil 20min then place on ice before use		*Herring testes carrier DNA. Boil 20min then place on ice before use
	Protocol		==Protocol==
	1. Grow 50mL overnight culture of yeast in YPDA or SD. Add several colonies to 1mL vortexing to break up clumps then pouring into 50 mL media in a 250 mL Erlenmeyer flask. Grow at 30C		#Grow 50mL overnight culture of yeast in YPDA or SD. Add several colonies to 1mL vortexing to break up clumps then pouring into 50 mL media in a 250 mL Erlenmeyer flask. Grow at 30C
	~~2. Transfer~~ overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3		#ransfer overnight culture to 300 mL YPDA to a OD600 of 0.2-0.3
	3. Incubate 3h at 30C to an OD of 0.4-0.6		#Incubate 3h at 30C to an OD of 0.4-0.6
	4. Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.		#Place Herring DNA on heating block at 95C for 20min then immediately on ice before use.
	5. Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min		#Pour cells into 50 mL Falcon tubes and centrifuge 1000g for 5min
	6. Discard supernatant and resuspend in 50 mL sterile TE or water		#Discard supernatant and resuspend in 50 mL sterile TE or water
	7. Centrifuge 1000g for 5min		#Centrifuge 1000g for 5min
	8. Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations		#Discard supernatant and resuspend in 1.5 mL of TE/LiAc. This is enough for about 14 transformations
	9. For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)		#For each cotransformation combine 0.2 ug Bait (pGBT) and 0.1 ug Prey (pGADGH) with 0.1 mg Herring DNA (10 uL)
	~~10. Add~~ 100 uL yeast cells and mix by vortexing		#dd 100 uL yeast cells and mix by vortexing
	~~11.~~ Add 0.6 mL PEG/LiAc solution and vortex to mix		#Add 0.6 mL PEG/LiAc solution and vortex to mix
	~~12.~~ Incubate at 30C for 30min with shaking		#Incubate at 30C for 30min with shaking
	~~13.~~ Add 70 uL DMSO		#Add 70 uL DMSO
	~~14.~~ Heat shock for 15min at 42C swirling occasionally to mix		#Heat shock for 15min at 42C swirling occasionally to mix
	~~15.~~ Chill on ice for 1-2 min		#Chill on ice for 1-2 min
	~~16.~~ Centrifuge at high speed for 5 s to pellet cells		#Centrifuge at high speed for 5 s to pellet cells
	~~17.~~ Resuspend in 0.5mL TE		#Resuspend in 0.5mL TE
	~~18.~~ Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media		#Plate 200 uL for cotransformation or 100 uL for single transformation onto appropriate selective media
	~~19.~~ Allow cells to grow for 3-5 days on selective media.		#Allow cells to grow for 3-5 days on selective media.

	[[Category:Yeast]]		[[Category:Yeast]]
	[[Category:Molecular Biology]]		[[Category:Molecular Biology]]
	[[Category:Transfection]]		[[Category:Transfection]]

Yeast Transfection (Small Scale) - Revision history

Davebrid: formatting changes

Davebrid: initial copy from protocol folder