IGEM:Peking/2007/Count:Conjugation - Revision history

Qu Mingzhi: /* Plating */

2007-09-01T06:23:45Z

Plating

←Older revision		Revision as of 06:23, 1 September 2007
Line 34:		Line 34:
	*Setting up serial dilutions:original, 10-1, 10-2, 10-3, 10-4, 10-5		*Setting up serial dilutions:original, 10-1, 10-2, 10-3, 10-4, 10-5
	*Pipette 200µL of each serial dilution onto an Double antibiotic plate.		*Pipette 200µL of each serial dilution onto an Double antibiotic plate.
		+	*Notes: Some controls may growth on double antibiotic plate when these cultures are not being diluted.

	==result==		==result==

Qu Mingzhi: /* Plating */

2007-09-01T06:13:52Z

Plating

←Older revision		Revision as of 06:13, 1 September 2007
Line 32:		Line 32:

	===Plating===		===Plating===
-	*Setting up serial dilutions:original, 10-1, 10-2.	+	*Setting up serial dilutions:original, 10-1, 10-2, 10-3, 10-4, 10-5
	*Pipette 200µL of each serial dilution onto an Double antibiotic plate.		*Pipette 200µL of each serial dilution onto an Double antibiotic plate.
		+
	==result==		==result==
	*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.		*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.
	==see also==		==see also==
	[[IGEM:UC_Berkeley/2006/Conjugation]]		[[IGEM:UC_Berkeley/2006/Conjugation]]

Qu Mingzhi at 11:21, 11 August 2007

2007-08-11T11:21:01Z

←Older revision		Revision as of 11:21, 11 August 2007
Line 34:		Line 34:
	*Setting up serial dilutions:original, 10-1, 10-2.		*Setting up serial dilutions:original, 10-1, 10-2.
	*Pipette 200µL of each serial dilution onto an Double antibiotic plate.		*Pipette 200µL of each serial dilution onto an Double antibiotic plate.
-	===result===	+	==result==
	*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.		*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.
	==see also==		==see also==
	[[IGEM:UC_Berkeley/2006/Conjugation]]		[[IGEM:UC_Berkeley/2006/Conjugation]]

Qu Mingzhi at 11:19, 11 August 2007

2007-08-11T11:19:31Z

←Older revision		Revision as of 11:19, 11 August 2007
Line 2:		Line 2:
	*choose the Donor cell and Recipient Cell which must be different Antibiotic.		*choose the Donor cell and Recipient Cell which must be different Antibiotic.

-	==*Day1:==	+	==Day1:==
	#Get the plates from -4 fridge：Donor, Recipient, Control.		#Get the plates from -4 fridge：Donor, Recipient, Control.
	#Amplification Culture in liquid LB for 12 hours.		#Amplification Culture in liquid LB for 12 hours.

-	==*day 2:==	+	==day 2:==
	#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.		#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.

Line 29:		Line 29:
	*Donor cells(log phase, include control) mixed with recipient cells(stationary phase).		*Donor cells(log phase, include control) mixed with recipient cells(stationary phase).
	*Mix by vortexing.		*Mix by vortexing.
-	*culture in 37℃ for 90 minutes,220rpm.	+	*culture in 37℃ for 90 minutes, 220rpm.

	===Plating===		===Plating===
-	*Setting up serial dilutions:	+	*Setting up serial dilutions:original, 10-1, 10-2.
-	*Pipette 200µL of each serial dilution onto an ~~SFM+MgCl2~~ plate	+	*Pipette 200µL of each serial dilution onto an Double antibiotic plate.
		+	===result===
		+	*The frequency of transfer was expressed as the ratio (percentage) of the number of transconjugants to that of the donors.
	==see also==		==see also==
	[[IGEM:UC_Berkeley/2006/Conjugation]]		[[IGEM:UC_Berkeley/2006/Conjugation]]

Qu Mingzhi: /* see also */

2007-08-11T11:15:02Z

Qu Mingzhi at 11:00, 11 August 2007

2007-08-11T11:00:29Z

←Older revision		Revision as of 11:00, 11 August 2007
Line 10:		Line 10:

	===Donor===		===Donor===
-	#Test OD600 after ~~30minutes~~ of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).	+	#Test OD600 after 30 minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
	#After sub-culturing decant the 500uL culture into a 1.5mL tube.		#After sub-culturing decant the 500uL culture into a 1.5mL tube.
	#spin down (top speed for 1 min), discard fluid.		#spin down (top speed for 1 min), discard fluid.
Line 26:		Line 26:
	#Place the cell suspension on ice.		#Place the cell suspension on ice.

-	===~~Plating the conjugant mix~~ ===	+	===mixing===
		+	*Donor cells(log phase, include control) mixed with recipient cells(stationary phase).
		+	*Mix by vortexing.
		+	*culture in 37℃ for 90 minutes,220rpm.

		+	===Plating===
		+	*Setting up serial dilutions:
		+	*Pipette 200µL of each serial dilution onto an SFM+MgCl2 plate
	==see also==		==see also==
	[[UC Berkeley/2006/Conjugation]]		[[UC Berkeley/2006/Conjugation]]

Qu Mingzhi at 10:39, 11 August 2007

2007-08-11T10:39:03Z

←Older revision		Revision as of 10:39, 11 August 2007
Line 1:		Line 1:
-	==Conjugation Test==	+	=Conjugation Test=
	*choose the Donor cell and Recipient Cell which must be different Antibiotic.		*choose the Donor cell and Recipient Cell which must be different Antibiotic.

-	~~'''~~*Day1:~~'''~~	+	==*Day1:==
-	#Get the plates from -4 ~~fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+)~~, ~~C600_R751~~, ~~Dh5α_psc101~~	+	#Get the plates from -4 fridge：Donor, Recipient, Control.
	#Amplification Culture in liquid LB for 12 hours.		#Amplification Culture in liquid LB for 12 hours.
-	~~'''~~*day 2:~~'''~~	+
		+	==*day 2:==
	#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.		#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
		+
	===Donor===		===Donor===
	#Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).		#Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
	#After sub-culturing decant the 500uL culture into a 1.5mL tube.		#After sub-culturing decant the 500uL culture into a 1.5mL tube.
-	#spin down (top speed for 1 min.), discard fluid.	+	#spin down (top speed for 1 min), discard fluid.
	#resuspend in 500mL LB(NO Antibiotic!), vortex.		#resuspend in 500mL LB(NO Antibiotic!), vortex.
	#Repeat steps 3-4-3 in this order then progress to step 6.		#Repeat steps 3-4-3 in this order then progress to step 6.
	#Re-suspend with 500mL LB.		#Re-suspend with 500mL LB.
	#Place the cell suspension on ice.		#Place the cell suspension on ice.
		+
	===Recipient===		===Recipient===
	#decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.		#decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
Line 25:		Line 28:
	===Plating the conjugant mix ===		===Plating the conjugant mix ===

-	==~~=result=~~==	+	==see also==
		+	[[UC Berkeley/2006/Conjugation]]

Qu Mingzhi: New page: ==Conjugation Test== choose the Donor cell and Recipient Cell which must be different Antibiotic. '''Day1:''' #Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+),...

2007-08-11T10:35:35Z

New page: ==Conjugation Test== *choose the Donor cell and Recipient Cell which must be different Antibiotic. '''*Day1:''' #Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+),...

New page

==Conjugation Test==
*choose the Donor cell and Recipient Cell which must be different Antibiotic.

'''*Day1:'''
#Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+), C600_R751, Dh5α_psc101
#Amplification Culture in liquid LB for 12 hours.
'''*day 2:'''
#put 2mL of culture into 20mL of LB with antibiotics, sub-culturing.
===Donor===
#Test OD600 after 30minutes of sub-culturing, stop subculturing when OD600 reached 0.45~0.6(log phase).
#After sub-culturing decant the 500uL culture into a 1.5mL tube.
#spin down (top speed for 1 min.), discard fluid.
#resuspend in 500mL LB(NO Antibiotic!), vortex.
#Repeat steps 3-4-3 in this order then progress to step 6.
#Re-suspend with 500mL LB.
#Place the cell suspension on ice.
===Recipient===
#decant the recipient cells(500uL culture into a 1.5mL tube) when reached the stationary phase.
#spin down (top speed for 1 min.), discard fluid.
#resuspend in 500mL LB(NO Antibiotic!), vortex.
#Repeat steps 3-4-3 in this order then progress to step 6.
#Re-suspend with 500mL LB.
#Place the cell suspension on ice.

===Plating the conjugant mix ===

===result===

←Older revision		Revision as of 11:15, 11 August 2007
Line 35:		Line 35:
	*Pipette 200µL of each serial dilution onto an SFM+MgCl2 plate		*Pipette 200µL of each serial dilution onto an SFM+MgCl2 plate
	==see also==		==see also==
-	[[~~UC Berkeley~~/2006/Conjugation]]	+	[[IGEM:UC_Berkeley/2006/Conjugation]]

IGEM:Peking/2007/Count:Conjugation - Revision history

Qu Mingzhi: /* Plating */

Qu Mingzhi: /* Plating */

Qu Mingzhi at 11:21, 11 August 2007

Qu Mingzhi at 11:19, 11 August 2007

Qu Mingzhi: /* see also */

Qu Mingzhi at 11:00, 11 August 2007

Qu Mingzhi at 10:39, 11 August 2007

Qu Mingzhi: New page: ==Conjugation Test== *choose the Donor cell and Recipient Cell which must be different Antibiotic. '''*Day1:''' #Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+),...

Qu Mingzhi: New page: ==Conjugation Test== choose the Donor cell and Recipient Cell which must be different Antibiotic. '''Day1:''' #Get the plates from -4 fridge：C600-R751_psc101(Tc+), Dh5α_psb1A2(Amp+),...