IGEM:UC Berkeley/2006/PCRPrep - Revision history

Jenlu: /* Culture PCR */

2006-09-13T17:32:29Z

Culture PCR

←Older revision		Revision as of 17:32, 13 September 2006
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	==Culture PCR==		==Culture PCR==
	#Grow colonies in 96 well blocks overnight		#Grow colonies in 96 well blocks overnight
-	#Spin the block in a swinging bucket centrifuge (full speed, 5-10 minutes) to pellet the cells	+	#Spin the block in a swinging bucket centrifuge (full speed~ 3500rpm, 5-10 minutes) to pellet the cells
	#Flick out the media		#Flick out the media
	#Resuspend the cells in the original media volume of water		#Resuspend the cells in the original media volume of water

2006-09-13T17:02:35Z

Culture PCR

←Older revision		Revision as of 17:02, 13 September 2006
Line 15:		Line 15:

	==Culture PCR==		==Culture PCR==
-	#Create PCR mixture.	+	#Grow colonies in 96 well blocks overnight
		+	#Spin the block in a swinging bucket centrifuge (full speed, 5-10 minutes) to pellet the cells
		+	#Flick out the media
		+	#Resuspend the cells in the original media volume of water
		+	#Create PCR mixture. Multiply each of the volumes below x the number of samples plus 1.
	#*20.8uL Water		#*20.8uL Water
	#*2.5uL or 10X thermol pol buffer		#*2.5uL or 10X thermol pol buffer
Line 23:		Line 27:
	#*0.5uL Reverse primer (10uM)		#*0.5uL Reverse primer (10uM)

-	#Add mixture to PCR tube.	+	#Add 24 uL of mixture to each PCR tube.
-	#~~Pick colony and add~~ to tube ~~and then streak an "X" on a plate while making a map of which colony is where.~~	+	#Add 1uL of resuspended cells to each PCR tube
	#On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.		#On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.

2006-09-13T16:59:46Z

←Older revision		Revision as of 16:59, 13 September 2006
Line 1:		Line 1:
	'''****Oligos are stored at 100 uM: dilute them to 10 uM*****'''		'''****Oligos are stored at 100 uM: dilute them to 10 uM*****'''

-	~~'''~~Colony PCR~~'''~~	+	==Colony PCR==
	#Create PCR mixture.		#Create PCR mixture.
-	#*21.8uL	+	#*21.8uL Water
	#*2.5uL or 10X thermol pol buffer		#*2.5uL or 10X thermol pol buffer
	#*0.5uL dNTPs (10mM)		#*0.5uL dNTPs (10mM)
Line 14:		Line 14:


-	~~'''~~PCR Prep (Extend Kit)~~'''~~	+	==Culture PCR==
		+	#Create PCR mixture.
		+	#*20.8uL Water
		+	#*2.5uL or 10X thermol pol buffer
		+	#*0.5uL dNTPs (10mM)
		+	#*0.1uL Taq
		+	#*0.5uL Forward primer (10uM)
		+	#*0.5uL Reverse primer (10uM)
		+
		+	#Add mixture to PCR tube.
		+	#Pick colony and add to tube and then streak an "X" on a plate while making a map of which colony is where.
		+	#On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.
		+
		+	==PCR Prep (Extend Kit)==

	# 36.25 ul water		# 36.25 ul water
Line 41:		Line 54:


-	~~'''~~PCR Prep (Phusion Kit)~~'''~~	+	==PCR Prep (Phusion Kit)==

	#Create PCR mixture		#Create PCR mixture

2006-07-20T19:06:08Z

2006-07-20T19:05:55Z

2006-07-17T20:04:28Z

←Older revision		Revision as of 20:04, 17 July 2006
Line 51:		Line 51:
	#*1uL DNA Template		#*1uL DNA Template
	#Run in PCR machine		#Run in PCR machine
		+
		+	Phusion polymerase adds at 1kbp/30sec so adjust 72C step accordingly. Also, if using the program 'JEDCOLONY' there is no need for a lysis step if you are PCRing from a miniprep. The 5min 92C step can be eliminated of shortened significantly.

	Keep polymerase on ice at all times		Keep polymerase on ice at all times

2006-07-07T18:27:00Z

←Older revision		Revision as of 18:27, 7 July 2006
Line 11:		Line 11:
	#Add mixture to PCR tube.		#Add mixture to PCR tube.
	#Pick colony and add to tube and then streak an "X" on a plate while making a map of which colony is where.		#Pick colony and add to tube and then streak an "X" on a plate while making a map of which colony is where.
-		+	#On the PCR machine use the "JED Colony" program and change the 72C cycle such that it matches the time needed to transcribe your product. Taq goes at about 1kb/minute.

2006-07-05T22:53:58Z

2006-07-05T22:53:33Z

←Older revision		Revision as of 22:53, 5 July 2006
Line 1:		Line 1:
		+	'''****Oligos are stored at 100 uM: dilute them to 10 uM*****'''
		+
	'''Colony PCR'''		'''Colony PCR'''
	#Create PCR mixture.		#Create PCR mixture.
Line 14:		Line 16:
	'''PCR Prep (Extend Kit)'''		'''PCR Prep (Extend Kit)'''

-	# 38.5 ul water	+	# 36.75 ul water
-	# 5 ul ~~buffer2~~	+	# 5 ul Expand tube "2" buffer
	# 5 ul dNTP		# 5 ul dNTP
-	# 1 ul oligo1	+	# 1 ul 10 uM oligo1
-	# 1 ul oligo2	+	# 1 ul 10 uM oligo2
	# 1 ul DNA template		# 1 ul DNA template
-	# .75 ul polymerase	+	# .75 ul Expand tube "1" polymerase

	50 ul total volume		50 ul total volume

		+
		+	Expand reagents are in Chris Anderson's -20 stuff<br>
	Combine in PCR tube.		Combine in PCR tube.

2006-06-29T18:33:33Z

←Older revision		Revision as of 18:33, 29 June 2006
Line 3:		Line 3:
	#*21.8uL		#*21.8uL
	#*2.5uL or 10X thermol pol buffer		#*2.5uL or 10X thermol pol buffer
-	#*0.5uL dNTPs (~~10uM~~)	+	#*0.5uL dNTPs (10mM)
	#*0.1uL Taq		#*0.1uL Taq
	#*0.5uL Forward primer (10uM)		#*0.5uL Forward primer (10uM)
Line 12:		Line 12:


-	'''PCR Prep'''	+	'''PCR Prep (Extend Kit)'''

	# 38.5 ul water		# 38.5 ul water
Line 35:		Line 35:

	-DLK_062106		-DLK_062106
		+
		+
		+	'''PCR Prep (Phusion Kit)'''
		+
		+	#Create PCR mixture
		+	#*10 uL 5x Buffer
		+	#*1uL dNTP (10mM)
		+	#*0.5uL Phusions (polymerase)
		+	#*1uL oligo 1 (10uL)
		+	#*1uL oligo 2 (10uL)
		+	#*1uL DNA Template
		+	#Run in PCR machine
		+
		+	Keep polymerase on ice at all times